Molecular cloning and expression of a fifth muscarinic acetylcholine receptor

C F Liao,A P Themmen,R Joho,C Barberis,M Birnbaumer,L Birnbaumer

doi:10.1016/s0021-9258(18)83237-9

C F Liao, A P Themmen + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)83237-9

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Abstract

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.

Highlights

From the Departmentsof $Cell Biology and 11Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030
Activation of signal transducingGproteins by GTPand thereby trigger cellular responses that depend on the affected G protein and the effector functions coupled by G proteins that areexpressed by the receiving cell or tissue [1,2]
Inorder to clone an M1-type muscarinicacetylcholine receptor (M1 receptor), we screened -250,000 recombinant XgtlO phages of a cDNA library constructed from rat brain centration to murine L cells expressing the open reading frame encoded in the BalIlDraI fragment of clone rgmb

Summary

Introduction

From the Departmentsof $Cell Biology and 11Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030. A cDNA of 2149 base pairs with an incomplete open ligand, nicotinic and muscarinic receptors are radically differreading frame(ORF)encoding amino acids 1-516 of a ent in structure and mode of action. 631-amino acid protein highly homologous to musca- multi-subunit, monovalent cationchannels that allow ion rinicreceptorswas cloned from a rat brain cDNA permeation and cause cell depolarization. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for thme uscarinic ligand [‘HI activation of signal transducingGproteins by GTPand thereby trigger cellular responses that depend on the affected G protein and the effector functions coupled by G proteins that areexpressed by the receiving cell or tissue [1,2]. Muscarinic stimulation of cells can cause intracellular Ca2+ mobilization due to activation of phospholipase C and formation of inositol trisphosphate, reduction of cAMP levels due to inhibition of adenylyl cyclase and/or stimulation of

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