Cysteine pairs in the third intracellular loop of the muscarinic m1 acetylcholine receptor play a role in agonist-induced internalization.
We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M(1) (hM(1)) receptor. Using site-directed mutagenesis, several mutant hM(1) receptors were made possessing either a deletion or point mutations within the third intracellular loop domain (252)PETPPGRCCRCC(263). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [(3)H]N-methylscopolamine (pK(D) = approximately 9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC(50) = approximately 5.5) relative to wild-type (pEC(50) = 5.35 +/- 0.05). In contrast, carbachol-induced internalization of mutant hM(1) receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM(1) receptor. Of the hM(1) receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM(1) receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM(1) receptors.
Sawyer
Gregory W
GW
Department of Biochemistry and Microbiology, Center for Health Sciences, Oklahoma State University, 1111 W. 17th Street, Tulsa, OK 74107-1898, USA. gwsawyer@chs.okstate.edu
Ehlert
Frederick J
FJ
Shults
Crystal A
CA
D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D019832:N-Methylscopolamine / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D017434:Protein Structure, Tertiary; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection
D010716:Phosphatidylinositols; D043643:Receptor, Muscarinic M1; D002217:Carbachol; D003545:Cysteine; D019832:N-Methylscopolamine