Oligomerization of G-protein-coupled Receptors Shown by Selective Co-immunoprecipitation

Jamil Bacha,Kamran Salim,Tim Fenton,Margaret Beer,Anne Heald,Julie Kerby,Emma Watts,George Mcallister,Hector Urien-Rodriguez,Paul C Guest,Heather A Skynner,Tim Bonnert

doi:10.1074/jbc.m201539200

Abstract

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.

Highlights

Recent studies have shown that G-protein-coupled receptors (GPCRs)1 may form dimers or higher order oligomers [1,2,3,4,5,6,7]
We have directly examined whether 5HT1 receptors are capable of forming oligomeric complexes with a variety of GPCRs by specific immunoprecipitation of each receptor followed by identification of the co-precipitated proteins using immunoblot analysis
Hetero-oligomerization of 5HT1A and 5HT1D Receptors with Other GPCRs— To directly test for hetero-oligomerization of different GPCR combinations we used sequential co-immunoprecipitation and immunoblot analyses of cells co-transfected with differentially epitope-tagged receptors

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases and other DNA-modifying enzymes were from New England Biolabs (Beverly, MA) or Amersham Biosciences. Construction of Epitope-tagged GPCRs and Transient Transfections—The c-Myc epitope (EQKLISEEDL) was inserted at the amino termini of 5HT1A and 5HT1D receptors by PCR mutagenesis using a Stratagene Robocycler with Hot-Top assembly (Amsterdam, Holland) as described by Nelson and Long [15]. Transient transfection of HEK-293 cells with the epitope-tagged constructs was performed by calcium phosphatemediated gene transfer as described previously [17]. The immunoadsorbents were recovered by centrifugation for 5 min at 700 ϫ g and washed three times by resuspension and centrifugation (5 min at 700 ϫ g) in cell lysis buffer and two times in 50 mM Tris (pH 7.5) containing 0.1% (w/v) SDS and 150 mM NaCl. The samples were eluted into 60 ␮l of SDS loading buffer (Sigma). Molecular size calibration was achieved with the MultiMark standards (Novex)

RESULTS

GPCR Oligomerization

DISCUSSION