Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Aug 16;91(17):7980–7984. doi: 10.1073/pnas.91.17.7980

Insertion mutagenesis as a tool to predict the secondary structure of a muscarinic receptor domain determining specificity of G-protein coupling.

K Blüml 1, E Mutschler 1, J Wess 1
PMCID: PMC44528  PMID: 8058746

Abstract

The N-terminal segment of the third intracellular loop (i3) of muscarinic acetylcholine receptors and other G protein-coupled receptors has been shown to largely determine the G-protein coupling selectivity displayed by a given receptor subtype. Based on secondary-structure prediction algorithms, we have tested the hypothesis that this region adopts an alpha-helical secondary structure. Using the rat m3 muscarinic receptor as a model system, a series of five mutant receptors, m3(+1A) to m3(+5A) were created in which one to five additional alanine residues were inserted between the end of the fifth transmembrane domain and the beginning of i3. We speculated that this manipulation should lead to a rotation of the N-terminal segment of the i3 domain (if it is in fact alpha-helically arranged), thus producing pronounced effects on receptor/G protein coupling. Pharmacological analysis of the various mutant receptors expressed in COS-7 cells showed that m3(+1A), m3(+3A), and m3(+4A) retained strong functional activity, whereas m3(+2A) and m3(+5A) proved to be virtually inactive. Helical wheel models show that this pattern is fully consistent with the notion that the N-terminal portion of i3 forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents the G-protein recognition surface.

Full text

PDF
7980

Images in this article

7983Image
on p.7983

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arden J. R., Nagata O., Shockley M. S., Philip M., Lameh J., Sadée W. Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor. Biochem Biophys Res Commun. 1992 Nov 16;188(3):1111–1115. doi: 10.1016/0006-291x(92)91346-r. [DOI] [PubMed] [Google Scholar]
  2. Baldwin J. M. The probable arrangement of the helices in G protein-coupled receptors. EMBO J. 1993 Apr;12(4):1693–1703. doi: 10.1002/j.1460-2075.1993.tb05814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berstein G., Blank J. L., Smrcka A. V., Higashijima T., Sternweis P. C., Exton J. H., Ross E. M. Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1. J Biol Chem. 1992 Apr 25;267(12):8081–8088. [PubMed] [Google Scholar]
  4. Blüml K., Mutschler E., Wess J. Functional role of a cytoplasmic aromatic amino acid in muscarinic receptor-mediated activation of phospholipase C. J Biol Chem. 1994 Apr 15;269(15):11537–11541. [PubMed] [Google Scholar]
  5. Blüml K., Mutschler E., Wess J. Identification of an intracellular tyrosine residue critical for muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis. J Biol Chem. 1994 Jan 7;269(1):402–405. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cheung A. H., Huang R. R., Graziano M. P., Strader C. D. Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the beta-adrenergic receptor. FEBS Lett. 1991 Feb 25;279(2):277–280. doi: 10.1016/0014-5793(91)80167-2. [DOI] [PubMed] [Google Scholar]
  9. Cheung A. H., Huang R. R., Strader C. D. Involvement of specific hydrophobic, but not hydrophilic, amino acids in the third intracellular loop of the beta-adrenergic receptor in the activation of Gs. Mol Pharmacol. 1992 Jun;41(6):1061–1065. [PubMed] [Google Scholar]
  10. Cotecchia S., Exum S., Caron M. G., Lefkowitz R. J. 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 Apr;87(8):2896–2900. doi: 10.1073/pnas.87.8.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cotecchia S., Ostrowski J., Kjelsberg M. A., Caron M. G., Lefkowitz R. J. Discrete amino acid sequences of the alpha 1-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis. J Biol Chem. 1992 Jan 25;267(3):1633–1639. [PubMed] [Google Scholar]
  12. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  13. DeLean A., Munson P. J., Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol. 1978 Aug;235(2):E97–102. doi: 10.1152/ajpendo.1978.235.2.E97. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Franke R. R., Sakmar T. P., Graham R. M., Khorana H. G. Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J Biol Chem. 1992 Jul 25;267(21):14767–14774. [PubMed] [Google Scholar]
  18. 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]
  19. Higashijima T., Burnier J., Ross E. M. Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J Biol Chem. 1990 Aug 25;265(24):14176–14186. [PubMed] [Google Scholar]
  20. Kunkel M. T., Peralta E. G. Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor. EMBO J. 1993 Oct;12(10):3809–3815. doi: 10.1002/j.1460-2075.1993.tb06059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lameh J., Philip M., Sharma Y. K., Moro O., Ramachandran J., Sadée W. Hm1 muscarinic cholinergic receptor internalization requires a domain in the third cytoplasmic loop. J Biol Chem. 1992 Jul 5;267(19):13406–13412. [PubMed] [Google Scholar]
  22. Lechleiter J., Hellmiss R., Duerson K., Ennulat D., David N., Clapham D., Peralta E. Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes. EMBO J. 1990 Dec;9(13):4381–4390. doi: 10.1002/j.1460-2075.1990.tb07888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  24. Parker E. M., Kameyama K., Higashijima T., Ross E. M. Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. J Biol Chem. 1991 Jan 5;266(1):519–527. [PubMed] [Google Scholar]
  25. Savarese T. M., Fraser C. M. In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J. 1992 Apr 1;283(Pt 1):1–19. doi: 10.1042/bj2830001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shapiro R. A., Nathanson N. M. Deletion analysis of the mouse m1 muscarinic acetylcholine receptor: effects on phosphoinositide metabolism and down-regulation. Biochemistry. 1989 Oct 31;28(22):8946–8950. doi: 10.1021/bi00448a039. [DOI] [PubMed] [Google Scholar]
  27. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  28. Strader C. D., Sigal I. S., Dixon R. A. Structural basis of beta-adrenergic receptor function. FASEB J. 1989 May;3(7):1825–1832. doi: 10.1096/fasebj.3.7.2541037. [DOI] [PubMed] [Google Scholar]
  29. Sukumar M., Higashijima T. G protein-bound conformation of mastoparan-X, a receptor-mimetic peptide. J Biol Chem. 1992 Oct 25;267(30):21421–21424. [PubMed] [Google Scholar]
  30. Voss T., Wallner E., Czernilofsky A. P., Freissmuth M. Amphipathic alpha-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins. J Biol Chem. 1993 Mar 5;268(7):4637–4642. [PubMed] [Google Scholar]
  31. 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]
  32. Wess J., Bonner T. I., Dörje F., Brann M. R. Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. Mol Pharmacol. 1990 Oct;38(4):517–523. [PubMed] [Google Scholar]
  33. Wess J., Brann M. R., Bonner T. I. Identification of a small intracellular region of the muscarinic m3 receptor as a determinant of selective coupling to PI turnover. FEBS Lett. 1989 Nov 20;258(1):133–136. doi: 10.1016/0014-5793(89)81633-3. [DOI] [PubMed] [Google Scholar]
  34. Wong S. K., Parker E. M., Ross E. M. Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists. J Biol Chem. 1990 Apr 15;265(11):6219–6224. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES