Homodimer Interface

While GPCR are generally conceptualized as forming dimeric/oligomeric complexes, very little is understood about the molecular interactions between dimers and oligomers and how these interactions regulate receptor function. Several different techniques have been used to explore the GPCR dimer interface, including peptides derived from various transmembrane domains (TMDs), immunoprecipitation, Western blot, cysteine cross-linking, mutagenesis, FRET, and molecular modeling. For class A GPCR, potential dimer interfaces have been reported to include the N-terminus of 8-opiod and yeast a-factor receptors (Cvejic and Devi 1997; Overton and Blumer 2002), TMD 1 of V2-vasopressin, C5a, a1b-adrenergic and rhodopsin receptors (Zhu and Wess 1998; Klco et al. 2003; Stanasila et al. 2003; Carrillo et al. 2004; Fotiadis et al. 2006), TMD 4 of C5a, rhodopsin, a1b-adrenergic and D2-dopamine receptors (Liang et al. 2003; Klco et al. 2003; Carrillo et al. 2004; Guo et al. 2005), TMD5 of rhodopsin receptors (Fotiadis et al. 2006), TMD 6 of b2-adrenergic and D2-dopamine receptors (Hebert et al. 1996; Ng et al. 1996), TMD 7 of a1b-adrenergic and D2-dopamine receptors (Stanasila et al. 2003; Ng et al. 1996), and the C-terminus of opiod receptors (Fan et al. 2005). The results of these experiments could be interpreted in several different ways: GPCR have multiple dimer interfaces to allow for oligomerization; members of the class A GPCR family use different mechanisms to form dimeric/oligomeric structures; or differences in the methods used to explore the dimer interface may yield different results. Clearly, additional studies designed to explore the GPCR dimer interface are warranted.

With respect to 5-HT2C receptors, Hendrickson and colleagues used a cysteine cross-linking approach in an attempt to elucidate potential dimer interfaces responsible for the formation of 5-HT2C homodimers (Mancia et al. 2008). In these studies, homology modeling based on the crystalline structure of rhodopsin provided a 5-HT2C receptor model that was used to identify residues with appropriate surface exposure for cysteine cross-linking. Candidate residues were mutated to cysteine, and transfected HEK 293 cells were treated with an oxidizing agent (or exposed to air); the formation of disulfide-linked dimers was evaluated by Western blot of detergent-solubilized cell lysates. These experiments yielded potential dimer interfaces at the extracellular end of TMD I (N55 and W56) and between TMD IV (I193) and TMD V (P213, N214). The putative TMD IV-V interface was sensitive to changes in receptor conformation following drug treatment. This observation is consistent with previous studies involving leukotriene B4, glutamate, and D2-dopamine receptors reporting conformational changes at the dimer interface during receptor activation and inactivation (Guo et al. 2005; Mesnier and Baneres 2004; Goudet et al. 2005). Previous studies have suggested that GPCR may use dimer interfaces at both TMD I and TMD IV-V to form oligomeric complexes (Milligan 2004; Fotiadis et al. 2006). However, higher-order oligomers of 5-HT2C receptors were not observed following cysteine cross-linking in cells coexpressing TMD I and TMD IV-V cysteine mutant receptors (Mancia et al. 2008). Based on these results and on experiments using receptor-Galpha fusion proteins, it was concluded that 5-HT2C receptor dimers are quasisymmetrical at the TMD IV-V interface and asymmetrical with respect to G-protein coupling (Mancia et al. 2008).

The results of the 5-HT2C receptor cysteine cross-linking experiments are consistent with prior reports of TMD I and TMD IV-V interfaces in dimer/oligomer models generated from the crystalline structure of rhodopsin. In contrast, the recently solved crystalline structure of the beta2-adrenergic receptor (b2-AR) reveals a very different type of dimeric interface (Cherezov et al. 2007): an interreceptor link between TMD1 and helix 8 (H8) of two adjacent receptors. To date, there have been no studies examining the validity of this model in vivo, and thus the functional significance remains unknown. A review of the literature concerning GPCR dimer interface studies reveals that TMD I and TMD IV-V are the most frequently cited regions for dimer/oligomer formation. Thus, it remains controversial as to whether there are two dimer interfaces, one involving TMD I and another involving TMD IV-V, leading to the formation of higher-order oligomeric structures or there is just a single dimer interface.

Studies using cross-linking reagents may be useful in distinguishing between these two different models. For example, Western blot experiments showed that the 5-HT2C receptor is sensitive to cross-linking with the membrane impermeable cross-linker BS3 (Herrick-Davis et al. 2004). Treatment with BS3 resulted in the appearance of immunoreactive bands the predicted size of dimers, but oligomers were not detected. There are only four lysine residues in the 5-HT2C receptor that are exposed to the extracellular environment such that they could participate in cross-linking following treatment of intact cells with BS3. There is one lysine residue in the N-terminus near the beginning of TMD I, one lysine residue in the extracellular loop (EL) II, and two lysine residues in the EL III. If a symmetrical or quasisymetrical TMD I dimer interface is responsible for the formation of 5-HT2C homodimers, then the lysine residue located in the N-terminus near the top of TMD I would likely be a key player in cross-linking with BS3. Therefore, a loss in cross-linking following mutation of this lysine residue would support a TMD I dimer interface model. On the other hand, a loss of cross-linking following mutation of the lysine residue in EL II (between TMD IV and V) would favor a model in which TMD IV-V were in close proximity to each other in the homodimer. Experimental design to minimize the potential for dimer capture would need to be considered, such as using very low receptor expression levels and very short cross-linking times.

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