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G-protein coupled receptor activation
Rhodopsin Dark State Conformation
- Crystal structure at 2.8A resolution (1F88.pdb) Palczewski, K., T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, and M. Miyano, Crystal structure of rhodopsin: A G protein-coupled receptor. Science, 2000. 289(5480): p. 739-45.
- further refinement at 2.8A resolution (1HZX.pdb) Teller, D.C., T. Okada, C.A. Behnke, K. Palczewski, and R.E. Stenkamp, Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry, 2001. 40(26): p. 7761-72
- new refinement at 2.6A resolution (1L9H.pdb): Okada, T., Y. Fujiyoshi, M. Silow, J. Navarro, E.M. Landau, and Y. Shichida, Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc Natl Acad Sci U S A, 2002. 99(9): p. 5982-7
- 7 water molecules, some of which are bound to highly conserved aa: Asn73/2.40; Asp83/2.50; Cys264/6.47; Asn302/7.49; Tyr306/7.53
- water cluster linked to Asn302, a key residue at the NPXXY motif in helix VII (highly conserved in rhodopsin-like GPCR), water cluster mediates interaction of side chain of Asn302 with residues in helices II, III, VI and VII y H-bonding network resulting in interhelical constraints. Disruption of these constraints is likely part of the activation process (i.e. moving apart of the helical bundle, e.g. Farrens et al., 1996; Sheikh et al, 1996). Water cluster includes Asp83 in helix II, which forms a direct H-bond to Asn55 in helix I, the N-D pair in rhodopsin-like GPCRs. Other residues involved in stabilizing and constraining the structure in this region around Asn55, Asp83 and Asn302 are Gly120, Met257, Ser298 and Tyr301.
- another set of water molecules important for color regulation
- the opening of the bundle in this area also demonstrated by antibody binding to Val304-Lys311 Abdulaev, N.G. and K.D. Ridge, Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin. Proc Natl Acad Sci U S A, 1998. 95(22): p. 12854-9.
- previous NMR and FTIR studies had predicted that the interaction between Schiff-base and counter-ion E113 is mediated by water molecule. However, there is no direct water link between Schiff base and carboxyl group of G113, but there is a water molecule in the vicinity which likely stabilizes the salt bridge by lowering the pKa of E113. That water is possibly liked to side chain of E113 via peptide amide of Cys187. There is a second water located between Glu181 and Ser186, close to the vicinity of E113 also.
- using microspectrophotometry it was shown that rhodopsin is functional in the crystal (dark state 495 nm, batho and lumirhodopsin and also MetaII are all observed)
Two-state versus multistate models of receptor activation:
-two-state = extended ternary complex model for ligand-induced activation: reviewed in Gether, U. and B. K. Kobilka (1998). "G protein-coupled receptors. II. Mechanism of agonist activation." J Biol Chem 273(29): 17979-82. and in Gether (2000) review
-the receptor exists in an equilibrium between inactive R and active R*
-in absence of agonist, inactive R is prevailing
-energy barrier between R and R* is sufficiently low, so that spontaneous conversion to R* occurs
-agonists are predicted to bind with highest affinity to R* and thereby shift the equilibrium towards R*
-inverse agonists (=negative antagonists) inhibit agonist-independent receptor activity, and stabilize the inactive R state,
-neutral antagonists bind with same affinity to R and R*
==> 2-state model not sufficient to explain complex behavior of GPCR, but rather there are multiple conformational states of GPCR - References:
1. Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.
2. Gether, U., J.A. Lowe, 3rd, and T.W. Schwartz, Tachykinin non-peptide antagonists: binding domain and molecular mode of action. Biochem Soc Trans, 1995. 23(1): p. 96-102.
3. Chidiac, P., T.E. Hebert, M. Valiquette, M. Dennis, and M. Bouvier, Inverse agonist activity of beta-adrenergic antagonists. Mol Pharmacol, 1994. 45(3): p. 490-9.
4. Riitano, D., T.M. Werge, and T. Costa, A mutation changes ligand selectivity and transmembrane signaling preference of the neurokinin-1 receptor. J Biol Chem, 1997. 272(12): p. 7646-55.
5. Reale, V., F. Hannan, L.M. Hall, and P.D. Evans, Agonist-specific coupling of a cloned Drosophila melanogaster D1-like dopamine receptor to multiple second messenger pathways by synthetic agonists. J Neurosci, 1997. 17(17): p. 6545-53.
6. Wiens, B.L., C.S. Nelson, and K.A. Neve, Contribution of serine residues to constitutive and agonist-induced signaling via the D2S dopamine receptor: evidence for multiple, agonist-specific active conformations. Mol Pharmacol, 1998. 54(2): p. 435-44.
7. Mhaouty-Kodja, S., L.S. Barak, A. Scheer, L. Abuin, D. Diviani, M.G. Caron, and S. Cotecchia, Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features. Mol Pharmacol, 1999. 55(2): p. 339-47.
- different constitutively active mutants of the alpha1B-receptor are differentially phosphorylated and internalized although they convey a similar agonist-independent activity
- direct structural evidence from fluorescence spectroscopy of purified beta2-adrenergic receptor, ref. Gether, U., J.A. Lowe, 3rd, and T.W. Schwartz, Tachykinin non-peptide antagonists: binding domain and molecular mode of action. Biochem Soc Trans, 1995. 23(1): p. 96-102.
- in rhodopsin, the less efficient activation of opsin by free trans retinal may more closely reflect the process of activation of other GPCR (Gether, 2000)
ternary complex model = cooperative interactions between GPCR, G protein and agonist
extended = include activation of G protein in the absence of agonists (constitutive activation)
also accounts for the effects of full agonists, partial agonists, natural antagonists and inverse agonists
in the extended model, the receptor exists in an equilibrium of the inactive and the activate state and the efficacy of ligands is a reflection of their ability to alter the equilibrium between these states
agonists: preferentially enrich active state
inverse agonists: preferentially enrich the inactive state
neutral antagonists: possess an equal affinity for both states and function simple as competitors
model may need to be further extended to account for more complicated relationships
activation of rhodopsin is unique among GPCR because ligand binding is not part of activation process
Activation kinetics:
- rhodopsin: activation microseconds, inactivation (Meta II decay) minutes
- beta2-adrenergic: activation 2-3 minutes, inactivation 30 sec
these slow kinetics cannot be explained by simple 2-state model, rather "sequential binding and conformational selection" model may be more appropriate, see Figure 5 in Gether, 2000
Qualitative studies of Conformational changes from rhodopsin to Meta II (see refs in Gether, 2000 review):
FTIR
SPR
tryptophan UV
EPR
A. Ligand-binding triggered activation via "aromatic cluster" in TM6
- Evidence from studies on Serotonin 5HT2AR receptor
"aromatic cluster" motif formed by conserved aromatic residues in TM6:
F6.44
W6.48: proposed to go from perpendicular to parallel (with respect to membrane) position as in rhodopsin (see below), but here upon binding of ligand to F6.52 (no experimental evidence for this conformational change though yet). This causes reduction of proline-kink of P6.50 (see section B, below).
F6.51
F6.52: serves as ligand sensor via interaction with ligand 5HT, triggers conformational change in W6.48
- Evidence from studies on rhodopsin
W6.48(265) goes from perpendicular to parallel orientation upon activation
F6.52 is NOT hydrophobic (in contradiction to "aromatic cluster" theory), instead P6.50(267) and W6.48(265) are conserved. However, W6.48 directly interacts with retinal, which keeps W6.48 in the perpendicular orientation. Here the sensor is 11-cis retinal (not like in 5HT receptor F6.52), which upon isomerization to all-trans releases constraint, and W6.48 can re-orient into its parallel position.
- Rotamer toggle switch in beta2-adrenergic receptor: Shi, L., G. Liapakis, R. Xu, F. Guarnieri, J.A. Ballesteros, and J.A. Javitch, Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. J Biol Chem, 2002. 277(43): p. 40989-96.
"agonist binding to a cluster of aromatic residues in TM VI may promote receptor activation by altering the configuration around the TM VI Proline kink and subsequent movement of CP end of TM VI away from TM III"
- biased Monte Carlo technique of conformational memories used to show that rotamer changes among Cys/Ser/Thr6.47, Trp 6.48 and Phe6.52 are highly correlated, representing a rotamer toggle switch that may modulate the Pro kink
- conformational switches in TM alpha-helices can be generated via Pro-containing motifs that form flexible molecular hinges: Sansom, M.S. and H. Weinstein, Hinges, swivels and switches: the role of prolines in signalling via transmembrane alpha-helices. Trends Pharmacol Sci, 2000. 21(11): p. 445-51
- although "rigid body" motion predicted, the conformational changes may involve flexibility about the hinge formed by the highly conserved Pro in TM VI (Pro288/6.50 in beta2-adrenergic receptor): Visiers, I., J.A. Ballesteros, and H. Weinstein, Three-dimensional representations of G protein-coupled receptor structures and mechanisms. Methods Enzymol, 2002. 343: p. 329-71.
- Cys6.47g+/Trp6.48t/Phe6.52t represents the active state and Cys6.47t/Trp6.48g+/Phe6.52g+ represents the inactive state
- with Trp 6.48 in t in the active state, Phe6.52 must also be in t to avoid steric clashes
- movement of Trp6.48 side chain from g+ to t upon activation is consistent with the inference from UV absorption spectrometry in rhodopsin that the indole side chain of Trp265/6.48 changes orientation during the inactive to active conformational transition [Lin, S.W. and T.P. Sakmar, Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry, 1996. 35(34): p. 11149-59] as well as with data suggesting that a single Trp tilts toward the membrane during activation [Chabre, M. and J. Breton, Photochem. Photobiol., 1979. 30: p. 295-299]
- photoisomerization of retinal from cis to trans moves the beta-ionone ring toward TM IV [Borhan, B., M.L. Souto, H. Imai, Y. Shichida, and K. Nakanishi, Movement of retinal along the visual transduction path. Science, 2000. 288(5474): p. 2209-12.] and thus away from Trp6.48, whose enhanced freedom and subsequent rearrangement is involved in activation of rhodopsin
- "the exquisite lack of constitutive activity of rhodposin may result from the inability of 6.48 to move to the t rotamer when constrained by bound 11-cis)
- for GPCR such as beta2-adrenergic receptor, a ligand that stabilizes Trp6.48 in the g+ conformation would therefore behave as an inverse agonist
- in the inactive state of rhodopsin, the side chain of Trp265/6.48 forms a H-bond with another residue [Lin, S.W. and T.P. Sakmar, Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry, 1996. 35(34): p. 11149-59]
- in the crystal structure, the only residue that can form a H-bond to indole of Trp265 is Cys264/6.47. Receptor activation alters tertiary interactions and weakens this H-bond. The Shi et al (2002) paper proposed that the interaction between Cys6.47 t and Trp6.48 g+ in the inactive state accounts for the H-bond feature of Trp265/6.48 that is lost during the activation process
- C6.47Thr is constitutively active
- Cys6.47 in t is positioned in distance to have H bond with its own Carbonyl, i-3 from Pro6.50 and may alter the Pro kink conformation thereby repositioning the CP end of TM VI away from TM III upon activation. Therefore , Cys6.47 through the interaction of its Sgamma with the indole Nepsilon of Trp6.48 and the H-bond of its side chain to its own carbonyl may liknk the conformation of the aromatic cluster with the conformation of the Pro-kink, thereby playing a role in receptor activation. This local network may underlie the observed correlations between the rotamer conformations of Cys6.47 and Trp6.48. The conformational changes will involve a change in interhelical interactions.
- activation also causes bulk dielectric changes surrounding Trp [Lin, S.W. and T.P. Sakmar, Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry, 1996. 35(34): p. 11149-59]
- Cys6.47 is unreactive in inactive state, and reactive to MTSEA in constitutively active mutant
- simulations performed on isolated TM VI out of the context of the helical bundle - may lead to artifacts
- summary: "hypothesis that coordinated rotamer changes of 6.47, 6.48 and 6.52 associated with receptor activation. This rotamer toggle switch may impact the alpha-helix backbone and may modulate the movement of TM VI about the Pro6.50-kink during activation. Cys6.47, Trp6.48 and Pro6.50 are highly conserved in rhodopsin-like receptors and 6.52 is always bulky (except in rhodopsin), it may be general for GPCR."
B. Helix Movement
Conformational changes of transmembrane helices are involved in receptor activation:
- Rhodopsin
Light-induced isomerization of 11-cis retinal causes a perturbation in the transmembrane domain that is transmitted to the cytoplasmic domain. The cytoplasmic domain changes its tertiary structure from the dark-state to a new structure, the light-activated state. The molecular details of this conformational change have been studied extensively using cysteine mutagenesis in conjunction with biochemical and biophysical studies.
Single Cysteines:
Yang et al.
Altenbach, Yang et al.
Ridge et al.
Farabaksh, Ridge et al.
Farabaksh et al.
Klein-Seetharaman et al. (1999) Biochemistry
Altenbach, Klein-Seetharaman et al. (1999) Biochemistry
Cai et al. (1999) Biochemistry
Altenbach, Cai et al. (1999) Biochemistry
Klein-Seetharaman et al. (1999) PNAS
Double Cysteines:
Farrens et al. (1996)
Klein-Seetharaman et al. (2001) Biochemistry
Altenbach, Klein-Seetharaman et al. (2001) Biochemistry
Cai et al. (2001) Biochemistry
Altenbach, Cai et al. (2001) Biochemistry
Zinc-Histidine:
Sheikh, S. P., T. A. Zvyaga, et al. (1996). "Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F." Nature 383(6598): 347-50.
The results from this approach have been reviewed in the following publication:
Figure: Helix movement upon activation of rhodopsin. Figure is Figure 1 in Meng and Bourne (2001) Trends in Pharmacological Sciences: Receptor activation: what does the rhodopsin structure tell us
- Dominant motion: outward and clockwise motion of TM6,
- Role of P6.50:
P6.50 is believed to enable activation-induced motion of TM6 flexible hinge at conserved P6.50,
P6.50 is located within the aromatic cluster (see Section A, above)
Changes in aromatic cluster cause a drastic decrease in the proline kink of TM6 (reduction in bend angle)
- Tryptophan UV show evidence for relative movement of TM III and VI. Mutation of Trp in TM III and VI eliminated the spectral differences in the UV absorbance spectra that distinguished rhodopsin from Meta II: Lin, S.W. and T.P. Sakmar, Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry, 1996. 35(34): p. 11149-59
- SPR of rhodopsin:
1. Salamon, Z., Y. Wang, J.L. Soulages, M.F. Brown, and G. Tollin, Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films. Biophys J, 1996. 71(1): p. 283-94.
2. Salamon, Z., Y. Wang, M.F. Brown, H.A. Macleod, and G. Tollin, Conformational changes in rhodopsin probed by surface plasmon resonance spectroscopy. Biochemistry, 1994. 33(46): p. 13706-11.
3. Salamon, Z., Y. Wang, G. Tollin, and H.A. Macleod, Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy. Biochim Biophys Acta, 1994. 1195(2): p. 267-75.
Other GPCR
Beta2-adrenergic receptor:
- movement of TM3 and TM6 on agonist activation with method below
Gether, U., S. Lin, and B.K. Kobilka, Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem, 1995. 270(47): p. 28268-75.
- derivatize cysteine with fluorescent label and then study effect of ligand binding on fluorescence- experiments show that not only agonists but also inverse agonists can promote structural changes in a GPCR
Gether, U., J.A. Ballesteros, R. Seifert, E. Sanders-Bush, H. Weinstein, and B.K. Kobilka, Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility. J Biol Chem, 1997. 272(5): p. 2587-90
IANBD labeling of Cys125 at end of helix III and Cys285 at end of helix VI undergo changes upon activation, exposed to more polar environment upon agonist binding
Cys285 becomes accessible to MTSEA in constitutively active mutant CAM, consistent with rotation and/or tilting of helix VI (same overall conclusion as in Rasmussen et al., 1999)
Jensen, A. D., F. Guarnieri, et al. (2001). "Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling." J Biol Chem 276(12): 9279-90. reviewed in : Jensen, A. D. and U. Gether (2000). "Assessing adrenergic receptor conformation using chemically reactive fluorescent probes." Methods Mol Biol 126: 345-61.
- IANBD at positions 6.31-34 at CP end of helix VI, purified
- upon agonist binding, all labels were in less polar environment
- only minor change in molecular environment for 6.31 and 6.32, but major change at 6.33 and 6.34
- supports movement of helix VI away for center of helical bundle and upwards
Sheikh et al., Zn-His studies indicate TM3/6 movement important for activation, see GPCR reviews
- Both Class A and Class B seem to be conserved: beta2-adrenergic and PTH receptors behave similarly: Sheikh, S.P., J.P. Vilardarga, T.J. Baranski, O. Lichtarge, T. Iiri, E.C. Meng, R.A. Nissenson, and H.R. Bourne, Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation. J Biol Chem, 1999. 274(24): p. 17033-41
- constitutively active mutants
- using cysteine accessibility in constitutively activated beta2-adrenergic receptor, a cysteine in TM VI became accessible in the binding crevice to a charged SH reagent, consistent with a counterclockwise rotation or tilting of the helix
- study of constitutively active mutant D130N (in D/E R Y motif) and in D130A,
- in D130N, a cysteine (C285) becomes accessible that is not accessible in the dark
- the position of that cysteine is consistent with movement of helix 6 like in rhodopsin
- propose that protonation leads to release of constraining intramolecular interactions
- see Section C below
- alpha1B-adrenergic receptor: Phe303 [ref. Chen, S., F. Lin, M. Xu, J. Hwa, and R.M. Graham, Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation. Embo J, 2000. 19(16): p. 4265-71.]
- homologous position in beta2-adrenergic receptor: F282 (towards CP end of helix VI, but not all the way)
- mutants at F282 are constitutively active: F282L > A> G
- substituted cysteine accessibility studies (SCAM) show that F282L causes movement of TM VI, both above and below (towards CP) a kink-inducing Pro (Pro288), whereas F282A is only below Pro288
- model in which rigid body motion of helix VI acts as a pivot because of its kink that can transduce and amplify agonist-induced conformation change in the EC/TM domain to result in a change in TM/CP domain
- alpha1B-adrenergic receptor:
Chen, S., F. Lin, M. Xu, J. Hwa, and R.M. Graham, Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation. Embo J, 2000. 19(16): p. 4265-71
- muscarinic receptor:
Spalding, T.A., E.S. Burstein, S.C. Henderson, K.R. Ducote, and M.R. Brann, Identification of a ligand-dependent switch within a muscarinic receptor. J Biol Chem, 1998. 273(34): p. 21563-8
- thyroid-stimulating hormone (TSH) receptor
Govaerts, C., A. Lefort, S. Costagliola, S.J. Wodak, J.A. Ballesteros, J. Van Sande, L. Pardo, and G. Vassart, A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor. J Biol Chem, 2001. 276(25): p. 22991-9.
- C5a receptor:
Baranski, T.J., P. Herzmark, O. Lichtarge, B.O. Gerber, J. Trueheart, E.C. Meng, T. Iiri, S.P. Sheikh, and H.R. Bourne, C5a receptor activation. Genetic identification of critical residues in four transmembrane helices. J Biol Chem, 1999. 274(22): p. 15757-65
- Dopamine D2 Receptor:
- cysteine accessibility of helix I (SCAM) with and without bound antagonist for Ala38/1.36 to Val59/1.57
- cysteine accessibility of the other helices, reviewed in:
1. Akabas, M.H., D.A. Stauffer, M. Xu, and A. Karlin, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science, 1992. 258(5080): p. 307-10.
2. Karlin, A. and M.H. Akabas, Substituted-cysteine accessibility method. Methods Enzymol, 1998. 293: p. 123-45. [ Also see all the Javitch et al. references in Xu et al (2001) Biochemistry paper]
- antagonist only protected N52/1.50C from reaction
- EC half of TM1 is not reactive except for A38/1.36C
- Pro1.48 bends the EC portion of TM 1 inward toward TM II and VII in opsins, but is absent in dopamine receptor, therefore helix I may be straighter and further away from helical bundle and not many conserved contact residues
- a model of dopamine receptor suggests that accessible residues in the CP half of helix I are at the interface with TM VII and H8
- model in which TM I, VII and H8 make contacts that stabilize the inactive state
[ note that the same thing is true in rhodopsin AB-loop-316 interactions, but that this constraint is not critical for activation - as shown by G-protein activation not being blocked by disulfide bond]
- they define their own measure of sequence similarity by calculating a value representing a combination of three measures, 1. the number of different amino acids, 2. the quantitative significance of pairwise substitution probabilities and 3. the frequency of appearance of each type of residue "conservation index"
- overall conservation index of the CP end of TM I and of EC end of TM I are similar in opsins, but in dopamine CP conservation is greater than EC conservation and equal to that in opsins
- N52/1.50C and V59/1.57C reacted more slowly than other cys at similar depth in the other TMs, suggesting dynamic structural fluctuations at the TM I-II-H8 interface
- mu-delta-kappa opioid receptors
- SCAM of TM VI in C7.38S background, based on study below
- about half of 22 mutants in each receptor showed inhibition of ligand binding by MTSEA - conclude which residues are on the water-accessible surface of the binding-site crevices
- at EC side, there is a wider area of accessibility, likely due to a proline kin at 6.50 which bends the helix toward the binding pocket and enables motion
- accessibility pattern is similar to that of Dopamine D2 receptor suggesting generality in class A
- ligand binding to C7.38S mutants was insensitive to MTSEA
- conserved C7.38 is differentially accessible in the binding-site crevice of the three receptor subtypes
- this paper forms basis for detailed SCAM study - see Xu et al 2001 Biochemistry paper
SCAM was developed by Karlin and Akabas to identify residues that line the channel of the nicotinic acetylcholine receptor [ Karlin, A. and M.H. Akabas, Substituted-cysteine accessibility method. Methods Enzymol, 1998. 293: p. 123-45.]
- used small charged methanethiosulfonate MTS reagents including MTS ethylammonium CH3SO2SCH2CH2NH3+ (MTSEA) to form mixed disulfide
- MTS react 10^9 times faster with ionized thiolates S- than with unionized thiols SH: [Roberts, D.D., S.D. Lewis, D.P. Ballou, S.T. Olson, and J.A. Shafer, Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry, 1986. 25(19): p. 5595-601.] and ionization is expected to occur to a significant extent only in the aqueous medium [ Karlin, A. and M.H. Akabas, Substituted-cysteine accessibility method. Methods Enzymol, 1998. 293: p. 123-45.]. Thus if binding of ligand is sensitive to reaction with MTS, we infer that the residue is on the water0accessible surface of the receptor. If this reaction is retarded by the presence of ligand, we further infer that the residue forms the surface of the binding-site crevice.
Characterization of complexity of conformational states and their dynamics by single molecule spectroscopy:
- use of single molecule spectroscopy: overcome the problem of ensemble averaging of other methods, ref.
1. Nie, S. and R.N. Zare, Optical detection of single molecules. Annu Rev Biophys Biomol Struct, 1997. 26: p. 567-96.
2. Nie, S., D.T. Chiu, and R.N. Zare, Probing individual molecules with confocal fluorescence microscopy. Science, 1994. 266(5187): p. 1018-21.
3. Dickson, R.M., A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature, 1997. 388(6640): p. 355-8.
4. Xie, X.S. and J.K. Trautman, Annu Rev Phys Chem, 1998. 49: p. 441-480.
- beta2-adrenergic receptor
- fluorescence tag in detergent micelles at position Cys265 (Fluorescein-5-maleimide) at the end of TM VI
- of 13 cysteines, Cys265 is the only accessible to the Fluorescein-5-maleimide (5 in TM region, 4 in disulfide bonds in EC domain, 1 in C-term is palmitoylated, remaining 2 C-terminal Cys form a disulfide bond during purification)
- at least two distinct substates for the native receptor, "is a conformation ally flexible molecule", 2 predominant, several minor ones possibly represented by different burst intensities
- full agonist ISO (which has higher binding affinity KI~10 microM and higher biological efficacy than adrenaline) stabilizes conformational substates that are rare in the native receptor
- conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes
- conformational heterogeneity of GPCR in the presence and absence of a bound agonist
- various ligands (agonists and antagonists) change both the shape of the entire distribution and the populations of the conformational substates
- conformational changes assessed by steady-state fluorescence spectroscopy of label at Cys265 (EF loop) - quenching by reagents in different environments
- fluorescence changes upon agonist binding: clockwise rotation of TM6 and/or tilting of CP end of TM6 towards TM5 --> similar to rhodopsin activation
- difference to rhodopsin: relatively slow kinetics of the conformational changes in beta2-AR which may reflect the different energetics of activation by diffusible ligands: rhodopsin activation in ms, while agonist activation of the beta2-AR is hundreds of seconds, although on-rate of agonist binding is fast (20s)
- no change in fluorescence intensity upon antagonist binding: either no change in conformation, or not detectable at that site
- strong correlation between reduction in fluorescence intensity and drug efficacy
- partial agonists: smaller change in fluorescence than full agonist: either receptor exists in 2 functional states or partial agonists induce a conformational change distinct from that induced by full agonists
=> conventional fluorescence spectroscopy (which represents an average intensity over a population) cannot distinguish these alternatives => therefore next publication:
- fluorescence lifetime spectroscopy: can detect discrete conformational states in a population of molecules whereas fluorescence intensity measurement reflect the weighted average of one or more discrete states
- fluorescence lifetime refers to the average time that a fluorophore that has absorbed a photon remains in the excited state before returning to the ground state
- the lifetime of fluorecein (ns) is much faster than the predicted off-rate of the agonists (microseconds-milliseconds) and much shorter than the half-life of conformational states of bR (microseconds), rhodopsin (milliseconds) or ion channels (micro-milliseconds) => thus, fluorescein can capture even short-lived conformational states
- fluorescence lifetime distributions can model the flexibility (local unfolding?) of protein structures, where the width of the distribution reflects the conformational flexibility of the protein
- can distinguish substates within a population of fluorescent molecules
- fluorescent labeled beta2-adrenergic receptor in CP domain (EF-loop: Cys265)
- in absence of ligands: oscillation around a single detectable conformation (Gaussian distribution of lifetimes centered at 4.2 ns), fluorescent decay rate modeled by a single exponential function
- binding of neutral antagonist: no change in the conformation, but reduction it the flexibility of the domain (detected by narrowing the distribution of lifetimes) [consistent with observation that receptor is less susceptible to protease digestion when bound to agonist)
- binding of full agonist: two distinct conformations
- previously shown that there is correlation between reduction in fluorescence intensity with drug efficacy => shortening the lifetime is associated with a reduction in fluorescence intensity => conclusion that the shorter lifetime is the active conformation
- binding of partial agonists: different conformations than when full agonist binds (indicated by different life-times)
- model for activation in which multiple agonist-specific receptor states exist, wherein activation occurs through a sequence of conformational changes:
Step 1. binding of agonist, the receptor undergoes a conformational change to an intermediate state that is associated with a narrowing in the long lifetime distribution
transitions from intermediate state to active state is rare high energy event
in vivo the active conformation is further stabilized by interaction of receptor and G protein
- Importance of Helix VII: an activating metal ion-binding site can be generated between TM III and VII (ref. Elling, C.E., S.M. Nielsen, and T.W. Schwartz, Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor. Nature, 1995. 374(6517): p. 74-7)
- also see nanotechnology
- agonist induced conformational changes as detected by surface plasmon resonance is slow (like for beta2-AR, see above)
- reason for being slow: because the elongation (that could be result of the helix movement and resulting in vertical movement of the CP loops) of the receptor also causes rearrangement of the lipid membrane (increase in positive curvature of the lipid surface)
- both agonist and antagonist increase the molecular orientational ordering of the receptor within the membrane
- both agonist and antagonist induce formation of several conformational states of the lipid membrane
- agonist: slow multiphasic kinetics indicate several conformational states involved in formation of final activated state as suggested by Gether and Kobilka (1998) (either equilibrium or preferential formation of one structure)
- significantly different structural changes induced upon binding of agonist versus antagonist: only agonist induces an increase in thickness and molecular packing density of the membrane, consistent with changes in the orientation of TM helices
- newly developed variant of surface plasmon resonance, called coupled plasmon-waveguide resonance CPWR spectroscopy:
- allows study of anisotropic membrane (and other nanostructure) systems
- real-time changes in structure of the receptor in parallel and perpendicular to the lipid membrane plane in resonance to ligand interactions
- femtomole amounts of receptor and ligand are needed
- measures mass density, conformation and molecular orientation changes
- utility of CPWR: general procedure to examine
1. ligand-receptor structural transitions
2. ligand-receptor interactions (including binding constants and time) - dose/response binding assay
3. minute amounts needed lend this technique to high-throughput screening
C. Consequences of the outward movement of helices for restraining contacts at CP ends of the helices: E/D R Y motif.
- mutate D -> constitutive activity; mutate R -> structural instability (but active conformation possible)
- conservation and pivotal role of DRY motif for signal transduction:
1. Oliveira, L., A.C. Paiva, and G. Vriend, A common motif in G-protein coupled seven transmembrane helix receptors. J Comp.-Aid. Mol. Des., 1993. 7: p. 649-658.
2. Zhu, S.Z., S.Z. Wang, J. Hu, and E.E. el-Fakahany, An arginine residue conserved in most G protein-coupled receptors is essential for the function of the m1 muscarinic receptor. Mol Pharmacol, 1994. 45(3): p. 517-23.
3. Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.
4. Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc Natl Acad Sci U S A, 1997. 94(3): p. 808-13.
5. Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84.
6. Ballesteros, J., S. Kitanovic, F. Guarnieri, P. Davies, B.J. Fromme, K. Konvicka, L. Chi, R.P. Millar, J.S. Davidson, H. Weinstein, and S.C. Sealfon, Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J Biol Chem, 1998. 273(17): p. 10445-53.
7. Probst, W.C., L.A. Snyder, D.I. Schuster, J. Brosius, and S.C. Sealfon, Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol, 1992. 11(1): p. 1-20.
8. Savarese, T.M. and C.M. Fraser, In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J, 1992. 283 ( Pt 1): p. 1-19.
- Arg is fully conserved, and mutation causes disease (Sung, C.H., C.M. Davenport, J.C. Hennessey, I.H. Maumenee, S.G. Jacobson, J.R. Heckenlively, R. Nowakowski, G. Fishman, P. Gouras, and J. Nathans, Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A, 1991. 88(15): p. 6481-5] and causes modest or severe impairment of signaling ability: Franke, R.R., T.P. Sakmar, R.M. Graham, and H.G. Khorana, Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J Biol Chem, 1992. 267(21): p. 14767-74; Acharya, S. and S.S. Karnik, Modulation of GDP release from transducin by the conserved Glu134-Arg135 sequence in rhodopsin. J Biol Chem, 1996. 271(41): p. 25406-11; Zhu, S.Z., S.Z. Wang, J. Hu, and E.E. el-Fakahany, An arginine residue conserved in most G protein-coupled receptors is essential for the function of the m1 muscarinic receptor. Mol Pharmacol, 1994. 45(3): p. 517-23.; Jones, P.G., C.A. Curtis, and E.C. Hulme, The function of a highly-conserved arginine residue in activation of the muscarinic M1 receptor. Eur J Pharmacol, 1995. 288(3): p. 251-7; Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.; Rosenthal, W., A. Antaramian, S. Gilbert, and M. Birnbaumer, Nephrogenic diabetes insipidus. A V2 vasopressin receptor unable to stimulate adenylyl cyclase. J Biol Chem, 1993. 268(18): p. 13030-3; Ballesteros, J., S. Kitanovic, F. Guarnieri, P. Davies, B.J. Fromme, K. Konvicka, L. Chi, R.P. Millar, J.S. Davidson, H. Weinstein, and S.C. Sealfon, Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J Biol Chem, 1998. 273(17): p. 10445-53.; Arora, K.K., Z. Cheng, and K.J. Catt, Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization. Mol Endocrinol, 1997. 11(9): p. 1203-12]
- Asp highly conserved, and mutation causes constitutive activity for some GPRCs [Cohen, G.B., T. Yang, P.R. Robinson, and D.D. Oprian, Constitutive activation of opsin: influence of charge at position 134 and size at position 296. Biochemistry, 1993. 32(23): p. 6111-5.; Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.; Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84.; Morin, D., N. Cotte, M.N. Balestre, B. Mouillac, M. Manning, C. Breton, and C. Barberis, The D136A mutation of the V2 vasopressin receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities. FEBS Lett, 1998. 441(3): p. 470-5; Acharya, S. and S.S. Karnik, Modulation of GDP release from transducin by the conserved Glu134-Arg135 sequence in rhodopsin. J Biol Chem, 1996. 271(41): p. 25406-11]
- receptor activation probably involves protonation
- in Histamine H2 receptor:
- histamine H2 receptor stably expressed in CHO cells is constitutively active at low expression levels 300fmol/mg protein
- D115 (in D/E RY motif) mutation --> constitutive activity
- R116 (in D/E R Y motif) mutation --> structurally unstable (expression only detectable when agonist or inverse agonist present); increased agonist affinity; signal transduction decreased --> conclusion: disruption of receptor stabilizing constraints leads to active conformations but also instability
- in 5HT2A-receptor:
- interaction between R3.50 and both, D3.49 and E6.30 ("ionic lock", "arginine cage") in the inactive state is broken by the helix movement (5HT2A-receptor)
- protonation state of carboxylic groups at D3.49 and E6.30 are believed to vary between active and inactive states of the receptors (5HT2A-receptor)
- computational modeling supports the interaction between R3.50 and both D3.49 and E6.30 contributing to close packing of the ends of helices III and VI (Visiers et al. (2002) Int. J. Quantum Chem. 88, 65-75) in 5HT2A-receptor:
- activation involves movement of helices VI and II apart, "but the molecular mechanisms involved in the helix rearrangements are not known"
- removal of acidic side-chain at position D3.49 or 6.30 or both in 5HT2A-receptor results in constitutive activity (see below) (Visiers et al (2002)):
Arg3.50 is caged in teh inactive state of the receptor by interaction with D3.49 and other conserved residues including E6.30 - thus D3.49/R3.50/E6.30 are a functional microdomain
- in opioid receptors
arginine cage involves R3.50 and T6.34D
- in beta2-adrenergic receptor
- D130 in D/E R Y motif mutated to N to mimic its protonation state and D130A to remove the functionality of the side chain
- both mutants are constitutively active
- in D130N, a cysteine in TM 6, Cys285, becomes accessible to methanethiosulfonate ethylammonium (charged sulfhydryl reagent)
- based on modeling, the position of Cys285 indicates that the D130N mutation has caused a counterclockwise rotation or tilting of TM6 (release of constraining intramolecular interactions)
- main reference for suggesting that ionic interactions between Asp;/Glu3.49, Arg3.50 and Glu6.40 may constitute a common switch governing the activation many rhodopsin-like receptors. Disruption of the interaction between TM III and TM VI produces constitutive activation and the extent of constitutive activation is highly correlated with the extent of conformational rearrangement in TM VI
- includes discussion of crystal structure
- probes ionic interactions between Arg3.50 and both Asp/Glu3.49 and Glu6.30 proposed to keep the CP ends of helices III and VI immobilized in the inactive state
- the previously proposed interaction of Arg3.50 with Asp2.50 in the polar pocket and arginine cage hypotheses is no longer supported by crystal structure, because these are 20A appart, and even upon light-activation are unlikely to move within interaction distance, therefore the new proposal is that Glu6.30 is the interaction partner and may be the second site of protonation upon activation (in contrast to previously proposed Glu3.28).
- in glycoprotein hormone receptors Glu6.30 mutant is constitutively active:
1. Parma, J., L. Duprez, J. Van Sande, R. Paschke, M. Tonacchera, J. Dumont, and G. Vassart, Constitutively active receptors as a disease-causing mechanism. Mol Cell Endocrinol, 1994. 100(1-2): p. 159-62.
2. Laue, L., W.Y. Chan, A.J. Hsueh, M. Kudo, S.Y. Hsu, S.M. Wu, L. Blomberg, and G.B. Cutler, Jr., Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty. Proc Natl Acad Sci U S A, 1995. 92(6): p. 1906-10.
3. Kosugi, S., T. Mori, and A. Shenker, An anionic residue at position 564 is important for maintaining the inactive conformation of the human lutropin/choriogonadotropin receptor. Mol Pharmacol, 1998. 53(5): p. 894-901.
- Glu/Asp 6.30 is not universally conserved among all Class A, but 100% in neurotransmitter, glycoportien homron and opsin receptors
- thus other interactions might serve the stabilization of inactive state function in the other receptors
- the originally constitutively active mutation in beta2-adrenergic receptor was actually in four position NEIGHBORING Glu6.30 (i.e. 6.28/6.29/6.31/6.34) and therefore these could contribute to the stabilization of inactive state [Samama, P., S. Cotecchia, T. Costa, and R.J. Lefkowitz, A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem, 1993. 268(7): p. 4625-36]
- Thr6.34 in rhodopsin crystal structure appears to contribut to the interaction wtih ARg3.50, and other constitutively active mutations Thr6.34 have been identified in amine GPCR: [
1. Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.
2. Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc Natl Acad Sci U S A, 1997. 94(3): p. 808-13.]
- instead of rigid body movement, it is proposed that activation involves flexibility around the hinge formed y the highly conserved proline in TM VI (Pro6.50) which causes TM VI to be highly bent with its CP end near the CP end of TM III
- activation may include straightening of the bent angle (IS THIS SUPPORTED BY BASAK/AJ results?)
- in beta1-adrenergic receptor:
- CP end of helix VI (C-III loop): L322I,T,E,F,C,A,K (=A293 in alpha1B; =L272 in beta2): constitutive activity strongest in L322K mutant [ref. Lattion, A., L. Abuin, M. Nenniger-Tosato, and S. Cotecchia, Constitutively active mutants of the beta1-adrenergic receptor. FEBS Lett, 1999. 457(3): p. 302-6]
- in alpha1B adrenergic receptor:
- R143K,H,E,D,A,N,I mutants have constitutive activity or are signaling impaired, but all have higher affinity to agonist (CP domain couples back to TM/EC domains!) and can drive isomerization of receptor into different conformations
D. Protonation
Rhodopsin - Arnis et al, 1994: proton uptake at E134
Beta2-adrenergic receptor - Ghanouni, P., H. Schambye, et al. (2000). "The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation." J Biol Chem 275(5): 3121-7.:
- homologous to E134 in rhodopsin is D142 in beta2-adrenergic receptor
- studied the effect of pH on ligand-induced activation of fluorescence-labeled purified beta2-AR
- at pH 6.5 2-fold increase in fluorescence change as compared to pH 8, although agonist affinity was lower at pH 6.5
- basal activity of beta2-AR is greater at pH 6.5 than at pH 8
E. Constitutive Activity
Constitutive activity observed with point mutations of residues located in all three receptor domains, EC, CP and TM:
Lefkowitz, R.J., S. Cotecchia, P. Samama, and T. Costa, Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci, 1993. 14(8): p. 303-7
Pauwels, P.J. and T. Wurch, Review: amino acid domains involved in constitutive activation of G-protein-coupled receptors. Mol Neurobiol, 1998. 17(1-3): p. 109-35
1. disruption of salt bridge between TM III and TM VII
rhodopsin:
Robinson, P.R., G.B. Cohen, E.A. Zhukovsky, and D.D. Oprian, Constitutively active mutants of rhodopsin. Neuron, 1992. 9(4): p. 719-25
alpha1B-adrenergic receptor:
Porter, J.E. and D.M. Perez, Characteristics for a salt-bridge switch mutation of the alpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity. J Biol Chem, 1999. 274(49): p. 34535-8
2. charge-neutralizing mutations of E/D in E/D R Y motif
Cohen, G.B., T. Yang, P.R. Robinson, and D.D. Oprian, Constitutive activation of opsin: influence of charge at position 134 and size at position 296. Biochemistry, 1993. 32(23): p. 6111-5.
Acharya, S. and S.S. Karnik, Modulation of GDP release from transducin by the conserved Glu134-Arg135 sequence in rhodopsin. J Biol Chem, 1996. 271(41): p. 25406-11
Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78
Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84
3. aa at CP end of helix VI
Javitch, J.A., D. Fu, G. Liapakis, and J. Chen, Constitutive activation of the beta2 adrenergic receptor alters the orientation of its sixth membrane-spanning segment. J Biol Chem, 1997. 272(30): p. 18546-9
4. aa further down in TM VI
- alpha1B-adrenergic receptor: Phe303 [ref. Chen, S., F. Lin, M. Xu, J. Hwa, and R.M. Graham, Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation. Embo J, 2000. 19(16): p. 4265-71.]
- homologous position in beta2-adrenergic receptor: F282 (towards CP end of helix VI, but not all the way)
- mutants at F282 are constitutively active: F282L > A> G
- substituted cysteine accessibility studies (SCAM) show that F282L causes movement of TM VI, both above and below (towards CP) a kink-inducing Pro (Pro288), whereas F282A is only below Pro288
- model in which rigid body motion of helix VI acts as a pivot because of its kink that can transduce and amplify agonist-induced conformation change in the EC/TM domain to result in a change in TM/CP domain
E6.30N,Q,L constitutively active in 5HT2A-receptor (Visiers et al (2002))
T6.34D inactive, T6.34K constitutively active in mu-opioid receptor (Huang et al. (2001) Biochemistry 40, 13501-13509)
T343R,S,K - T343R is constitituively active in dopamine D2 receptor [Wilson, J., H. Lin, D. Fu, J.A. Javitch, and P.G. Strange, Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation. J Neurochem, 2001. 77(2): p. 493-504]
Systematic screen that selects ACTIVE receptor mutants of the C5a receptor Baranski, T. J., P. Herzmark, et al. (1999). "C5a receptor activation. Genetic identification of critical residues in four transmembrane helices." J Biol Chem 274(22): 15757-65.:
- screen of random mutagenesis identified 121 functional mutants in 93 amino acid sites in TM helices 3,5,6 and 7 out of 523 mutants
- 21 constitutively active mutants
- looked for physical properties of side chains that were preserved in functional receptors (based on log odd score of 1 or great in PAM250 matrix)
- some positions of high importance and conservation in GPCR are sites of frequent substitution in functional C5a receptor mutants
- constitutively active mutants only found in helices 3 and 6, not in helices 5 and 7
- high tolerance of helix 6 for amino acid substitutions (more than 2x higher rates than in other helices)
- susceptibility of helix 6 to activating mutations is shared by biogenic amine, luteinizing hormone and thyrotropin receptors
Figure taken from Baranski et al: Side
chains are preserved on faces of helices that point toward other helices in the
Baldwin model. Helical wheel representations are based on the Baldwin model
(16). Helices are presented as viewed from the cytoplasm and
in positions that correspond to their relative positions at the middle of the
bilayer (16). Larger circles indicate residues closer
to the cytoplasm. Red letters and numbers indicate residues and
positions at which side chain character is preserved in mutant functional
receptors (see text). Dark blue circles indicate positions at which
hydrophobicity is preserved; white indicates positions that tolerate
polar substitutions; other positions are gray. Blue, orange,
and green letters indicate positions (Ile-124, Leu-127, and Phe-251,
respectively) at which substitutions caused constitutive activation of the
receptor.
activation mechanism by C5a receptor: Gerber et al., 2001
- site-directed mutagenesis of binding pocket shows differential effects on agonist and antagonist binding
- proposed mechanism: agonists induce a change in the relative orientations of helices 3 and 7 that allows movement of helix 6 aw from the bundle
- the ligand C5a must interact with TM domain, but can also interact with N-terminus (but this latter interaction is not required, only increases ligand affinity)
- impression that comes through from paper is that activation is solely based on TM domain, and that CP and EC domains are just there for affinity changes, not for the switch itself [this is not consistent with coupling of the three domains], also see quote here
F. Interconversion of different conformations of receptors
Class A peptide receptors, e.g. NK-1 receptor:
- mutations can affect the ability of the receptor to feely interchange between distinct receptor conformations, which bind the non-peptide antagonist and peptide agonist with high affinity, respectively, ref.
Rosenkilde, M.M., M. Cahir, U. Gether, S.A. Hjorth, and T.W. Schwartz, Mutations along transmembrane segment II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding. J Biol Chem, 1994. 269(45): p. 28160-4
- clearly different receptor conformational states that display distinct selectivity for the tachykinin peptides (NK-1 receptor): ref.
Ciucci, A., C. Palma, S. Manzini, and T.M. Werge, Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide. Br J Pharmacol, 1998. 125(2): p. 393-401.
Ciucci, A., C. Palma, D. Riitano, S. Manzini, and T.M. Werge, Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation. FEBS Lett, 1997. 416(3): p. 335-8
- similar observations in kappa-opioid receptor , ref.
Hjorth, S.A., K. Thirstrup, and T.W. Schwartz, Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor. Mol Pharmacol, 1996. 50(4): p. 977-84
G. Arguments against Generality Principle of Mechanisms of Activation
- "the binding modes for agonists is as diverse as the chemical nature of the ligands: even agonists at the same receptor mayu not necessarily share an overlapping binding site" (Gether, 2000)
- "it seems clear that there are multiple ways of propagating activation of GPCR" (Gether, 2000)
- "there is no common "lock" for all agonists' "keys"" (Gether, 2000), quotes ref. Schwartz, T.W. and M.M. Rosenkilde, Is there a 'lock' for all agonist 'keys' in 7TM receptors? Trends Pharmacol Sci, 1996. 17(6): p. 213-6
- evidence against generality: receptor-activating antibodies directed against EC loop of alpha1 and beta1-adrenergic receptors, in serum from patients with malignant hypertension and idiopathic dilated dilated cardiomyopathy, shows that even when ligands bind in TM domain normally, there is an alternative way of activating the respective receptors
- other activating antibodies:
muscarinic receptor
bradykinin B2 receptor
autoantibody against EC domains of TSH receptor in Grave's disease
==> multiple ways of activating GPCR. However, "the underlying fundamental mechanisms of activation" are likely to be conserved
H. GPCR are kept silent by constraining intramolecular interactions (see Gether review, 2000)
- many GPCR have basal activity and can activate the G protein in the absence of agonists
- mutations can increase the constitutive agonist-independent receptor activity (see above)
- initially constitutive activating mutants identified in C-III of adrenergic receptors, but now there are mutations all over the place, even in the EC domain, e.g. E-II of TSH receptor and E-III of thrombin receptor
- chimera between E-II in beta2/alpha1B-adrenergic receptor is constitutively active
- constitutive activity linked to diseases:
TSH receptor - hereditary thyroid adenomas
LH receptor - male precocious puberty
rhodopsin - RP
- molecular mechanism of constitutive activity: Ala293 VI.0/6.34 in C-terminal part of C-III of alpha1B-adrenergic receptor was substituted by all 20 aa, and all have higher basal activity (Lefkowitz et al: Kjelsberg, M.A., S. Cotecchia, J. Ostrowski, M.G. Caron, and R.J. Lefkowitz, Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem, 1992. 267(3): p. 1430-3.) ==> constraining intramolecular interaction have been conserved to keep receptor in an inactive conformation in the absence of agonist and these inactivating constraints could be released as a part of the receptor activation mechanism, either after agonist binding of due to specific mutations, causing key sequences to be exposed to the G protein
- hypothesis supported by dynamics:
constitutively activated beta2-adrenergic receptor mutation are characterized by a marked structural instability and enhanced conformational flexibility of the purified receptor proteins, refs.:
Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84
Gether, U., J.A. Ballesteros, R. Seifert, E. Sanders-Bush, H. Weinstein, and B.K. Kobilka, Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility. J Biol Chem, 1997. 272(5): p. 2587-90
==> the mutations have disrupted intramolecular interactions allowing the receptor to undergo conversion more readily between the inactive and active state
- using chimeric LH/FSH receptors it was shown that stabilizing interaction between TM V and VI are important for resistance of FSH receptor to constitutively activating mutations
- stabilizing role of TM VI in random mutagenesis study of muscarinic M5 receptor, where mutants on one side of helix confer constitutive activity
- mutation of polar residue sin TM VI of alpha-factor pheromone receptor (STE2p) cause constitutive activity
- molecular modeling of LH receptor also show helix VI helix packing important to keep receptor in an inactive configuration
- in rhodopsin
Met257, VI.05/6.40 in TM VI and NPXXY motif in TM VII
salt bridge Lys296 VII.10/7.43 and Glu113 III.04/3.28
- in angiotensin receptor
Asn111 III.11/3.35 and Tyr292(VII.10/7.43)
- in alpha1b-adrenergic receptor
Asp125 III.08/3.32 and Lys331 VII.03/7.36
- random mutagenesis study in muscarinic rececptor coupling: Burstein, E.S., T.A. Spalding, and M.R. Brann, The second intracellular loop of the m5 muscarinic receptor is the switch which enables G-protein coupling. J Biol Chem, 1998. 273(38): p. 24322-7:
in C-II substitutions on one side of alpha-helix III extending into CP side cause constitutive activity, while on opposite side of the helix they impair G protein activation (see also results by Yang et al., 1995, where he showed greater than 100% G protein activation for some mutants!) - first aa are important for keeping receptor in inactive conformation, the other for activation
the AspIII.25/3.49 of the D/E R Y motif is on the same side of the helix as the residues causing constitutive activation
- study of constitutively active mutant D130N (in D/E R Y motif) and in D130A,
- in D130N, a cysteine (C285) becomes accessible that is not accessible in the dark
- the position of that cysteine is consistent with movement of helix 6 like in rhodopsin
- propose that protonation leads to release of constraining intramolecular interactions
- emphasizes the CP ends of TM III and VI as critical switch region controlling activation of GPCR (beta2 adrenergic receptor)
I. Protonation is a key element in GPCR activation
How is disruption of stabilizing intramolecular interactions (see section H. above) initiated after agonist binding?
One event is protonation of D/E R Y "protonation hypothesis" - evidence:
charge neutralization causes dramatic constitutive activation in beta2-adrenergic receptor, see Gether (2000) review
GnRH similar
M1 muscarinic receptor similar
rhodopsin
- experiment similar to that Jong/Wayne with rhodopsin had been carried out with beta2-adrenergic receptor (Ref. Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84):
Asp130 III.25/3.49 mutation to Asn not only activates but also causes a cysteine in TM VI (CYs285 VI.12/6.47) to be accessible to methanethiosulfonate ethylammonium (MTSEA) a chareged reagent. This is consistent with counter clockwise rotation (as seen from EC side) or tilting of TM VI in the mutant
- 2 computational studies to define the role of protonation:
1. "Polar Pocket" hypothesis
Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78
ab initio Molecular Dynamics protocol (start with TM domain, then CP and EC domains; CHARMM, SHAKE algorithm, 1ns simulations) studies of alpha1B adrenergic receptor and comparison to constitutively active mutants D142A and A293E
the invariably conserved R143=ArgIII.26/3.50 is constrained in a pocket of H-bonding network formed by conserved polar residues in TM I, II and VII, including N63=AsnI.18/1.50, D91=AspII.10/2.50, N344=Asn VII.16/7.49 and Y348=TyrVII.19/7.52
upon protonation of AspIII.25/3.49, Arg shifts out of the pocket leading to long-range conformational changes in the receptor
ionic counterpart of Arg was conserved AspII.10/2.50, and this specific contact is broken upon activation
further studies of Polar Pocket hypothesis by experiment: R143K,H,E,D,A,N,I mutants of alpha1b adrenergic receptor (Scheer, A., T. Costa, F. Fanelli, P.G. De Benedetti, S. Mhaouty-Kodja, L. Abuin, M. Nenniger-Tosato, and S. Cotecchia, Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation. Mol Pharmacol, 2000. 57(2): p. 219-31):
increased affinity of all mutants for agonist
R143K is constitutively active, R143A,N,I,E are essentially dead
correlation between affinity shift and activity of the agonists
conclusion: R143 mutations drive isomerization of alpha1b into different conformations (i.e. coupling of CP domain back to EC/TM domains)
Results of the modeling for WT :
in D142A and A293E
D91-R143 interaction is disrupted
rearrangement of CP domains by the opening of a solvent exposed site by the i2 and the CP ends of V and VI and 242-259 forming an alpha-helical segment in i3 loop
--> large solvent accessible surface is formed, with good electrostatic and shape complementarity with the G protein
- cytosolic exposure of cationic residues (R288, K291) which are important for signaling
in R143K mutant:
arrangement with similarities to that of the constitutively active D142A and A293E mutants, i.e. exposure of R288 and K291 and the exposed opening
the shorter side chain of Lys does not favor the interaction with D91, thus mimicking the breakage of D91-R143 interaction in the A293E and D142A mutants
in R143A,N,I,E mutants:
do not permit the translocation of R288 and K291 towards cytosol
in D142A/R143A and A293E/R143A mutants:
none of the above changes
BUT:
apparent polymorphism of the phenotype which can be associated with mutations of E/DRY motif in different GPCR
Example: in rhodopsin, D83N does not impair the transition to Meta II and D83 is protonated both in dark and MetaII and is only weakly H-bonded in Meta II, therefore does not seem to play a major role either as acceptor or donor in the active state of rhodopsin (in contrast to polar pocket and arginine cage hypothesis)
2. "Arginine Cage" hypothesis
from studies in GnRH receptor (Monte Carlo simulation on the isolated helxi III, not the entire receptor model)
the ionic counterpart of Arg is AspIII.25/3.49, not AspII.10/2.50 as in the Polar Pocket hypothesis
upon activation, Arg will interact with AspII.10/2.50 (so the roles of that Arg in helix II are switched in the two models)
support for this hypothesis comes from rhodopsin experiment: AspII.10/2.50 (D83 in rhodopsin) is more strongly hydrogen bonded upon activation, ref. Rath, P., L.L. DeCaluwe, P.H. Bovee-Geurts, W.J. DeGrip, and K.J. Rothschild, Fourier transform infrared difference spectroscopy of rhodopsin mutants: light activation of rhodopsin causes hydrogen-bonding change in residue aspartic acid-83 during meta II formation. Biochemistry, 1993. 32(39): p. 10277-82
Renewed support for Arginine cage hypothesis: Huang, P., J. Li, C. Chen, I. Visiers, H. Weinstein, and L.Y. Liu-Chen, Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp. Biochemistry, 2001. 40(45): p. 13501-9.
- X1BBX2X3B motif at CP end of helix VI (B=basic; X=non-basic) mutations are constitutively active by releasing constraint between D/ERY and helix VI:
shown in rhodopsin, alpha1B-adrenergic, beta2-adrenergic, m1 muscarinic, 5-HT2A, 5-HT2C, 5-HT1B and cannabinoid CB1 receptors - refs:
1. Samama, P., S. Cotecchia, T. Costa, and R.J. Lefkowitz, A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem, 1993. 268(7): p. 4625-36.
2. Cotecchia, S., S. Exum, M.G. Caron, and R.J. Lefkowitz, Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc Natl Acad Sci U S A, 1990. 87(8): p. 2896-900.
3. Kjelsberg, M.A., S. Cotecchia, J. Ostrowski, M.G. Caron, and R.J. Lefkowitz, Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem, 1992. 267(3): p. 1430-3.
4. Ren, Q., H. Kurose, R.J. Lefkowitz, and S. Cotecchia, Constitutively active mutants of the alpha 2-adrenergic receptor. J Biol Chem, 1993. 268(22): p. 16483-7.
5. Lattion, A., L. Abuin, M. Nenniger-Tosato, and S. Cotecchia, Constitutively active mutants of the beta1-adrenergic receptor. FEBS Lett, 1999. 457(3): p. 302-6.
6. Hogger, P., M.S. Shockley, J. Lameh, and W. Sadee, Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors. J Biol Chem, 1995. 270(13): p. 7405-10.
7. Egan, C., K. Herrick-Davis, and M. Teitler, Creation of a constitutively activated state of the 5-HT2A receptor by site-directed mutagenesis: revelation of inverse agonist activity of antagonists. Ann N Y Acad Sci, 1998. 861: p. 136-9.
8. Herrick-Davis, K., C. Egan, and M. Teitler, Activating mutations of the serotonin 5-HT2C receptor. J Neurochem, 1997. 69(3): p. 1138-44.
9. Pauwels, P.J., A. Gouble, and T. Wurch, Activation of constitutive 5-hydroxytryptamine(1B) receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its G(o)alpha protein interactions. Biochem J, 1999. 343 Pt 2: p. 435-42.
10. Abadji, V., J.M. Lucas-Lenard, C. Chin, and D.A. Kendall, Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs. J Neurochem, 1999. 72(5): p. 2032-8.
- in this paper, mutations of X3 in mu-opioid receptor T6.34(279) K,D: K disrupts interaction, D strengthens interaction
- they built a homology model of alpha1b-adrenergic receptor based on crystal structure of rhodopsin
- constraint between D/E R Y and CP end of helix VI:
salt bridge between R143/3.50 of D/E R Y motif and E289/6.30 on CP end of helix VI constrains the receptor in the inactive state: mutations that weaken the interaction constitutively activate the receptor. Also investigated role of F286/6.27, A292/6.33, L296/6.37, V299/6.40, V300/6.41 and F303/6.44 on the interaction
- rationalized mutants in view of the model
Constitutive activity of mGluR
- reviewed in Hermans and Challis (2001) review
- review of GPCR in general: de Ligt, R.A., A.P.
Kourounakis, and I.J. AP, Inverse agonism
at G protein-coupled receptors: (patho)physiological relevance and implications
for drug discovery. Br J Pharmacol, 2000. 130(1):
p. 1-12
- mGlu1a but not mGlu1b,c in HEK293 cells exhibit constitutive activity with respect to phosphoinositide turnover [ Prezeau, L., J. Gomeza, S. Ahern, S. Mary, T. Galvez, J. Bockaert, and J.P. Pin, Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity. Mol Pharmacol, 1996. 49(3): p. 422-9]
- constitutive activity can be suppressed by MPEP [Pagano, A., D. Ruegg, S. Litschig, N. Stoehr, C. Stierlin, M. Heinrich, P. Floersheim, L. Prezeau, F. Carroll, J.P. Pin, A. Cambria, I. Vranesic, P.J. Flor, F. Gasparini, and R. Kuhn, The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. J Biol Chem, 2000. 275(43): p. 33750-8.)
- in neuronal cells, the constitutive activity of mGlu1/5 is normally suppressed through interactions with cytoskeletal elements, i.e. homer proteins [Ango, F., L. Prezeau, T. Muller, J.C. Tu, B. Xiao, P.F. Worley, J.P. Pin, J. Bockaert, and L. Fagni, Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer. Nature, 2001. 411(6840): p. 962-5]
- some evidence for requirement for extracellular Ca2+ in receptor ligand interactions, supported by crystal structure which contains metal binding site [Kunishima, N., Y. Shimada, Y. Tsuji, T. Sato, M. Yamamoto, T. Kumasaka, S. Nakanishi, H. Jingami, and K. Morikawa, Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature, 2000. 407(6807): p. 971-7.]