Bradykinin-induced Internalization of the Human B2Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail

doi:10.1074/jbc.274.18.12738

Bradykinin-induced Internalization of the Human B₂Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail*

From ^‡INSERM Unité 367, 17 rue du Fer àMoulin 75005 Paris, France and the ^¶Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Federal Republic of Germany

Abstract

The binding of bradykinin (BK) to B₂ receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B₂ receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor’s cytoplasmic tail corresponding to positions 321–364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A₂. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335–351, as well as alanine substitution of serine and threonine residues within segment 335–351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of β-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.

Footnotes

↵* This work was supported by INSERM and grants from the Bristol-Meyers Squibb Institute for Medical Research (Princeton, NJ) and the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by a grant from La Fondation Pour la Recherche Medicale. To whom correspondence should be addressed. Tel.: 33-1-45-87-61-00; Fax: 33-1-45-35-66-29; E-mail: pizard{at}ifm.inserm.fr.
↵‖ Present address: Ludwig Institute for Cancer Research, P.O. Box 595, S-75124 Uppsala, Sweden.
↵2 A. Faussner and A. Blaukat, unpublished observations.
Abbreviations:

GPCRs

G protein-coupled receptors

HBSS

Hank’s buffered saline solution

BK

bradykinin

B₂wt

human B₂ BK wild-type receptor

COSB₂wt

COS-7 cells overexpressing the B₂wt

PLC

phospholipase C

PLA₂

phospholipase A₂

BSA

bovine serum albumin

IPs

inositol phosphates

[Ca²⁺]_i

intracellular Ca²⁺

CHO

Chinese hamster ovary

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

PAGE

polyacrylamide gel electrophoresis
- Received August 12, 1998.
- Revision received January 21, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.