Structural Implication for Receptor Oligomerization from Functional Reconstitution Studies of Mutant V2 Vasopressin Receptors*
- From the Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, D-14195 Berlin, Germany
Abstract
Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1–3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.
Footnotes
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↵* This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.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.
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↵‡ To whom correspondence should be addressed: Inst. für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany. Tel.: 49-30-8445-1861; Fax: 49-30-8445-1818; E-mail: schoberg@zedat.fu-berlin.de.
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↵2 A. Schulz and T. Schöneberg, unpublished data.
- Abbreviations:
- GPCR
-
G-protein-coupled receptor
- AVP
-
arginine vasopressin
- ELISA
-
enzyme-linked immunosorbent assay
- HA
-
hemagglutinin
- PBS
-
phosphate-buffered saline
- TMD
-
transmembrane domain
- V2-R
-
V2 vasopressin receptor
- CCKA-R
-
cholecystokinin type A receptor
- m3-R
-
rat m3 muscarinic receptor
- GFP
-
green fluorescent protein
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- Received June 2, 1999.
- Revision received October 14, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
