Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.
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.
Wess
J
J
National Institute of Neurological Disorders and Stroke, Laboratory of Molecular Biology, Bethesda, MD 20892.
Nanavati
S
S
Vogel
Z
Z
Maggio
R
R
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002217:Carbachol / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003062:Codon / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019832:N-Methylscopolamine; D010276:Parasympatholytics / Q000378:metabolism / Q000494:pharmacology; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D011392:Proline; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012602:Scopolamine Derivatives / Q000378:metabolism; D014162:Transfection; D014364:Tryptophan
D003062:Codon; D010276:Parasympatholytics; D010880:Piperidines; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012602:Scopolamine Derivatives; D012313:RNA; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D014364:Tryptophan; D002217:Carbachol; D011392:Proline; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D019832:N-Methylscopolamine