Ligand-induced Phosphorylation/Dephosphorylation of the Endogenous Bradykinin B2 Receptor from Human Fibroblasts

doi:10.1074/jbc.271.50.32366

Ligand-induced Phosphorylation/Dephosphorylation of the Endogenous Bradykinin B2 Receptor from Human Fibroblasts*

From the ^¶ Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Federal Republic of Germany and the
^∥ Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Federal Republic of Germany

^″ To whom correspondence should be addressed. Tel.: 49-6131-395890; Fax: 49-6131-395793; E-mail: werner.mulleresterl{at}uni-mainz.de

Abstract

We have studied the ligand-induced phosphorylation/dephosphorylation of the bradykinin B2 receptor endogenously expressed in human HF-15 fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36), derived from the extreme carboxyl terminus of the human B2 receptor, precipitated the receptor from solubilized membranes of HF-15 cells that had been labeled with [³²P]orthophosphate. A low basal level of B2 receptor phosphorylation was found in the absence of a ligand. Stimulation of the cells with the B2 receptor agonists bradykinin, [Lys⁰,Hyp³]bradykinin, kallidin, and T-kinin resulted in a rapid and efficient phosphorylation of the receptor. The B2 receptor antagonist HOE140 and the B1 receptor agonist des-Arg⁹-bradykinin failed to induce significant phosphorylation of the B2 receptor. Phosphoamino acid analysis revealed that the B2 receptor is phosphorylated on serine and threonine, but not on tyrosine residues. The ligand-induced phosphorylation of the receptor was concentration-dependent, with an apparent EC₅₀ of 33 nM, and peaked at 1 min after challenge. The kinin-stimulated phosphorylation of the B2 receptor was rapid and transient and paralleled the kinetics of desensitization/resensitization of the receptor as followed by [Ca²⁺]_i release and radioligand binding assay, respectively. The ligand-induced phosphorylation of the B2 receptor was independent of the protein kinase C pathway. In vitro experiments suggest βARK1 (-drenergic eceptor inase) as a candidate kinase that could mediate the homologous B2 receptor phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively blocked the dephosphorylation, but did not affect the internalization of the B2 receptor, whereas inhibitors of receptor internalization delayed its dephosphorylation. These finding point to a role of ligand-induced phosphorylation in the desensitization and redistribution of the bradykinin receptor in human fibroblasts.

Footnotes

↵* This work was supported in part by Grant Mu 598/3-2 from the Deutsche Forschungsgemeinschaft, Grant BMH4-CT95-1008 from the European Commission, and a grant from the Fonds der Chemischen Industrie (to W. M.-E.). 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.
↵¹ The abbreviations used are:

DMEM

Dulbecco's modified Eagle's medium

PMA

phorbol 12-myristate 13-acetate

cAMP-S

adenosine cyclic 3′:5′-phosphorothioate

ID

intracellular domain

ED

extracellular domain

PIPES

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

MEM

minimum essential medium

PAGE

polyacrylamide gel electrophoresis.
↵² A. Blaukat and W. Müller-Esterl, unpublished results.
- Received July 8, 1996.
- Revision received September 11, 1996.