Instability of a Class A G Protein-Coupled Receptor Oligomer Interface

Abstract

The quaternary structure of G protein-coupled receptors (GPCRs) can influence their trafficking and ability to transduce signals. GPCR oligomers are generally portrayed as long-lived entities, although the stability of these complexes has not been studied. Here we show that D2 dopamine receptor protomers interact transiently at a specific oligomer interface. Selective immobilization of cyan fluorescent protein-D2 receptors (CD2Rs) in the plasma membrane failed to completely immobilize coexpressed D2-venus receptors (D2R-Vs), suggesting that the two did not form stable oligomers with each other. Oxidative cross-linking stabilized C-D2R-D2R-V oligomers such that immobilization of C-D2R also immobilized D2R-V. This stabilization required the presence in both C-D2R and D2R-V of a cysteine residue in transmembrane domain 4 (TM4), a region identified as a putative oligomer interface in these and other class A GPCRs. These results suggest that the interaction of D2 receptor protomers at TM4 is transient unless stabilized and that the quaternary structure of these receptors may thus be subject to physiological or pharmacological regulation.GPCRs can exist and function in cells as dimers or higherorder oligomers (Bouvier, 2001;Milligan, 2007). However, the structural arrangement of GPCR protomers within oligomers and the dynamics of oligomer assembly and disassembly are not well understood (Gurevich and Gurevich, 2008). The most is known about class C (metabotropic glutamate-like) oligomers; the interfaces between class C protomers involve either extracellular disulfide bonds or intracellular coiled-coils and are believed to be relatively stable (Bouvier, 2001). By comparison, the interfaces between class A (rhodopsin-like) protomers are poorly defined, although it is likely that transmembrane regions are involved. Transmembrane class A interfaces identified by molecular models and crystallography occupy relatively small protein surfaces (Liang et al., 2003;Lodowski et al., 2007), suggesting that oligomers formed at these interfaces might not be stable. However, it is difficult to predict class A GPCR oligomer stability because these interfaces are buried in a hydrophobic environment and because the proteins are concentrated in a two-dimensional membrane.To directly assess the stability of a class A GPCR oligomer interface, we selectively immobilized a subset of protomers on the surface of cells using specific antibodies. The lateral mobility of non-cross-linked protomers was then measured using fluorescence recovery after photobleaching (FRAP). The mobility of non-cross-linked protomers should have been decreased if these protomers formed stable oligomers with antibody-cross-linked protomers. We chose to study D2 dopamine receptors because oxidative cross-linking studies have identified the fourth transmembrane helix (TM4) of this receptor as part of a conformationally sensitive homo-oligomer interface (Guo et al., 2003(Guo et al., , 2005. TM4 (together with TM5) has also been identified as an interface between ...

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