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Overexpression and Functional Characterization of Kinin Receptors Reveal Subtype-Specific Phosphorylation†,‡

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Institute of Physiological Chemistry and Pathobiochemistry, University of Mainz, Duesbergweg 6, D-55099 Mainz, Germany, and Acadia Pharmaceuticals Incorporated, 3911 Sorrento Valley Boulevard, San Diego, California 92121
Cite this: Biochemistry 1999, 38, 4, 1300–1309
Publication Date (Web):January 5, 1999
https://doi.org/10.1021/bi981727r
Copyright © 1999 American Chemical Society
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    Abstract

    G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.

    †

     This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (MU 598/4-3) and from the Fonds der Chemischen Industrie (to W.M.E.).

    ‡

     This work forms part of the thesis of K.H.

    §

     University of Mainz.

    ‖

     Both authors contributed equally to this study.

    ⊥

     Present address:  Ludwig Institute for Cancer Research, Biomedical Center, Box 595, Husargatan 3, S-75124 Uppsala, Sweden.

    #

     Acadia Pharmaceuticals Inc.

    *

     To whom correspondence should be addressed. Phone:  +49−6131−395890. Fax:  +49−6131−395793. E-mail:  [email protected].

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