Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling

doi:10.1074/jbc.271.3.1507

Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling

RECONSTITUTION OF THE BINDING SITE BY CO-EXPRESSION OF TWO DEFICIENT MUTANTS (*)

From the From INSERM, Unité 36, College de France, 3, rue d'Ulm, 75005 Paris, France

^§ To whom correspondence should be addressed. Tel.: 33-1-44-27-16-75; Fax: 33-1-44-27-16-91.

Abstract

Type 1 angiotensin receptors (AT) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser, Ser, and Ser are not essential for maintenance of the angiotensin II binding site. Replacement of Asn or Ser does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT (DuP753)- or AT (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser mutant suggests that this residue, in addition to previously identified residues, Asp and Tyr, participates in the receptor activation mechanism.

Finally, Lys (third helix) and Lys (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.

Footnotes

↵^¶ A Foreign Maître de Conférence Associé professor at the College de France.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¹ The abbreviations used are:

AngII

angiotensin II

AT receptor

type 1 angiotensin II receptor

AT receptor

type 2 angiotensin II receptor

G-protein

guanine nucleotide-binding protein

PLC

phospholipase C

IP

inositol trisphosphate

IP

inositol phosphate

TM

transmembrane domain

CHO

Chinese hamster ovary

PCR

polymerase chain reaction

RT-PCR

reverse transcription PCR.
↵²P. Broto, unpublished results.
- Received March 20, 1995.
- Revision received October 6, 1995.