Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies

doi:10.1074/jbc.271.3.1748

Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies (*)

From the (1)Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Germany, the
(2)Institute Jacques Monod, Université Paris VII, Place Jussieu, F75005 Paris, France, and the
(3)Biotechnology Unit, Syntex Inc., Palo Alto, California 94304

^§ To whom correspondence should be addressed:
Inst. of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Germany
. Tel.: 49-6131-395890; Fax 49-6131-395792; muellere{at}mzdmza.zdv.unimainz.deainz.de.

Abstract

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa. Antibodies to extracellular domain 4 (third loop) blocked binding of the agonist but not of the antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding. Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding. Further, affinity-purified antibodies to the amino-terminal portion of extracellular domain 3 transiently increased intracellular free Ca concentration and thus are agonists. The Ca signal was specifically blocked by the B2 antagonist HOE140. By contrast, antibodies to the carboxyl-terminal segment of extracellular domain 4 failed to trigger Ca release. The specific effects of antibodies to the amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in agonist function.

Footnotes

↵* This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Mu 598/4-2) and the Fonds der Chemischen Industrie(163323) (to W. M. E.). 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.

This paper is dedicated to Dr. Hans Fritz on the occasion of his 60th birthday.
↵¹ The abbreviations used are:

WGA

wheat germ agglutinin

CHO

Chinese hamster ovary

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

E64

N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin

ED

extracellular domain

fura-2/AM

{1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid, pentaacetoxymethylester}

HMEM

minimum essential medium buffered with 20 mM Na-HEPES, pH 7.4, 1.8 mM Ca

HPP-HOE140

3-(4-hydroxyphenyl-propionyl)-HOE140

ID

intracellular domain

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PIPES

piperazine-N, N′-bis(2-ethanesulfonic acid)

TM

transmembrane segment.
↵²The domain designation originally proposed for the vasopressin V2 receptor is used(35).
↵³A. Miller, K. Jarnagin, S. Bhakta, C. Bach, C. Yee, T. Ho, T. Pan, R. Tahilramani, J. H. B. Pease, and R. Freedman, manuscript in preparation.
- Received August 2, 1995.
- Revision received October 2, 1995.