Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

E Kurtenbach,C.A Curtis,E.K Pedder,A Aitken,A.C Harris,E.C Hulme

doi:10.1016/s0021-9258(18)77406-1

Abstract

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

Highlights

From the Division of Physical Biochemistry Hill, London NW7 IAA, United Kingdom and Laboratory of Protein
The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned ml Muscarinic acetylcholine receptors (mAChR)
Products-We have shown previously that mAChRs purified from rat forebrain are labeled near-stoichiometrically by [3H]

Summary

Introduction

From the Division of Physical Biochemistry Hill, London NW7 IAA, United Kingdom and Laboratory of Protein. Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [‘Hlpropylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]iV-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. Ml mAChRs were purified from CHO-Kl cells stably expressing the cloned receptor sequence and labeled with [‘H]PrBCM. Two major CNBr cleavage products were found with a molecular mass of -3.9 and -2.4 kDa, labeled either by [‘HI. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned ml mAChR. The results were compatible with the attachment of the t3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, ml sequence) and of [3H]NEM to a conserved cysteine residue

Objectives

Results

Conclusion