A conserved motif in the membrane proximal C-terminal tail of human muscarinic m1 acetylcholine receptors affects plasma membrane expression.
We investigated the functional role of a conserved motif, F(x)(6)LL, in the membrane proximal C-tail of the human muscarinic M(1) (hM(1)) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence (423)FRDTFRLLL(431). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [(3)H]N-methylscopolamine binding assays. The plasma membrane expression of hM(1) receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM(1) receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L(430) and L(431) play a role in folding hM(1) receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V(46) and L(47), that, when mutated, reduce the plasma membrane expression of hM(1) receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM(1) receptors to achieve a transport-competent state.
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. gregory.sawyer@okstate.edu
Ehlert
Frederick J
FJ
Shults
Crystal A
CA
D020816:Amino Acid Motifs; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D017510:Protein Folding; D043643:Receptor, Muscarinic M1 / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D043643:Receptor, Muscarinic M1; D001285:Atropine; D002217:Carbachol