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. 1993 Jan;12(1):331–338. doi: 10.1002/j.1460-2075.1993.tb05661.x

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

J Wess 1, S Nanavati 1, Z Vogel 1, R Maggio 1
PMCID: PMC413210  PMID: 7679072

Abstract

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.

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Selected References

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  1. Berridge M. J., Dawson R. M., Downes C. P., Heslop J. P., Irvine R. F. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J. 1983 May 15;212(2):473–482. doi: 10.1042/bj2120473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonner T. I., Buckley N. J., Young A. C., Brann M. R. Identification of a family of muscarinic acetylcholine receptor genes. Science. 1987 Jul 31;237(4814):527–532. doi: 10.1126/science.3037705. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  8. Curtis C. A., Wheatley M., Bansal S., Birdsall N. J., Eveleigh P., Pedder E. K., Poyner D., Hulme E. C. Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor. J Biol Chem. 1989 Jan 5;264(1):489–495. [PubMed] [Google Scholar]
  9. Dixon R. A., Sigal I. S., Candelore M. R., Register R. B., Scattergood W., Rands E., Strader C. D. Structural features required for ligand binding to the beta-adrenergic receptor. EMBO J. 1987 Nov;6(11):3269–3275. doi: 10.1002/j.1460-2075.1987.tb02645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dohlman H. G., Caron M. G., DeBlasi A., Frielle T., Lefkowitz R. J. Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor. Biochemistry. 1990 Mar 6;29(9):2335–2342. doi: 10.1021/bi00461a018. [DOI] [PubMed] [Google Scholar]
  11. Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
  12. Dörje F., Wess J., Lambrecht G., Tacke R., Mutschler E., Brann M. R. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther. 1991 Feb;256(2):727–733. [PubMed] [Google Scholar]
  13. Findlay J. B., Pappin D. J. The opsin family of proteins. Biochem J. 1986 Sep 15;238(3):625–642. doi: 10.1042/bj2380625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Findlay J., Eliopoulos E. Three-dimensional modelling of G protein-linked receptors. Trends Pharmacol Sci. 1990 Dec;11(12):492–499. doi: 10.1016/0165-6147(90)90050-i. [DOI] [PubMed] [Google Scholar]
  15. Fraser C. M., Chung F. Z., Wang C. D., Venter J. C. Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5478–5482. doi: 10.1073/pnas.85.15.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fraser C. M. Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation. J Biol Chem. 1989 Jun 5;264(16):9266–9270. [PubMed] [Google Scholar]
  17. Fraser C. M., Wang C. D., Robinson D. A., Gocayne J. D., Venter J. C. Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function. Mol Pharmacol. 1989 Dec;36(6):840–847. [PubMed] [Google Scholar]
  18. Hargrave P. A., McDowell J. H. Rhodopsin and phototransduction: a model system for G protein-linked receptors. FASEB J. 1992 Mar;6(6):2323–2331. doi: 10.1096/fasebj.6.6.1544542. [DOI] [PubMed] [Google Scholar]
  19. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  20. Hibert M. F., Trumpp-Kallmeyer S., Bruinvels A., Hoflack J. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol Pharmacol. 1991 Jul;40(1):8–15. [PubMed] [Google Scholar]
  21. Hulme E. C., Birdsall N. J., Buckley N. J. Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol. 1990;30:633–673. doi: 10.1146/annurev.pa.30.040190.003221. [DOI] [PubMed] [Google Scholar]
  22. Hulme E. C., Kurtenbach E., Curtis C. A. Muscarinic acetylcholine receptors: structure and function. Biochem Soc Trans. 1991 Feb;19(1):133–138. doi: 10.1042/bst0190133. [DOI] [PubMed] [Google Scholar]
  23. Kurtenbach E., Curtis C. A., Pedder E. K., Aitken A., Harris A. C., Hulme E. C. Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation. J Biol Chem. 1990 Aug 15;265(23):13702–13708. [PubMed] [Google Scholar]
  24. Larrivee D. C., Conrad S. K., Stephenson R. S., Pak W. L. Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster. J Gen Physiol. 1981 Nov;78(5):521–545. doi: 10.1085/jgp.78.5.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakayama T. A., Khorana H. G. Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin. J Biol Chem. 1991 Mar 5;266(7):4269–4275. [PubMed] [Google Scholar]
  26. Nakayama T. A., Khorana H. G. Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal. J Biol Chem. 1990 Sep 15;265(26):15762–15769. [PubMed] [Google Scholar]
  27. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  28. Probst W. C., Snyder L. A., Schuster D. I., Brosius J., Sealfon S. C. Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 1992 Jan-Feb;11(1):1–20. doi: 10.1089/dna.1992.11.1. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shapiro R. A., Scherer N. M., Habecker B. A., Subers E. M., Nathanson N. M. Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene. J Biol Chem. 1988 Dec 5;263(34):18397–18403. [PubMed] [Google Scholar]
  31. Shieh B. H., Stamnes M. A., Seavello S., Harris G. L., Zuker C. S. The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein. Nature. 1989 Mar 2;338(6210):67–70. doi: 10.1038/338067a0. [DOI] [PubMed] [Google Scholar]
  32. Strader C. D., Sigal I. S., Candelore M. R., Rands E., Hill W. S., Dixon R. A. Conserved aspartic acid residues 79 and 113 of the beta-adrenergic receptor have different roles in receptor function. J Biol Chem. 1988 Jul 25;263(21):10267–10271. [PubMed] [Google Scholar]
  33. Strader C. D., Sigal I. S., Register R. B., Candelore M. R., Rands E., Dixon R. A. Identification of residues required for ligand binding to the beta-adrenergic receptor. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4384–4388. doi: 10.1073/pnas.84.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Strosberg A. D. Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins. Eur J Biochem. 1991 Feb 26;196(1):1–10. doi: 10.1111/j.1432-1033.1991.tb15778.x. [DOI] [PubMed] [Google Scholar]
  35. Wess J., Bonner T. I., Brann M. R. Chimeric m2/m3 muscarinic receptors: role of carboxyl terminal receptor domains in selectivity of ligand binding and coupling to phosphoinositide hydrolysis. Mol Pharmacol. 1990 Dec;38(6):872–877. [PubMed] [Google Scholar]
  36. Wess J., Gdula D., Brann M. R. Site-directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding. EMBO J. 1991 Dec;10(12):3729–3734. doi: 10.1002/j.1460-2075.1991.tb04941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van Koppen C. J., Nathanson N. M. Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function. J Biol Chem. 1990 Dec 5;265(34):20887–20892. [PubMed] [Google Scholar]

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