The dominant negative effect of receptor mutants on the ability of the wildtype receptor to traffic to the cell surface when co-expressed indicates that a GPCR oligomer must be in an appropriate conformation within the ER to permit cell-surface expression. For other membrane-spanning proteins such as the potassium ion channels, oligomerization is well-established to be important for proper cell-surface expression (57). In this case, correct oligomeriza-tion of ion channel subunits results in the formation of a fully functional ion channel within the ER that is trafficked to the cell surface. Proper oligomeric formations may facilitate cell-surface expression by masking ER retention signals (such as the RXR motif ) and exposing export motifs (such as the DXE motif ) found on ion channel subunits. These retention and retrieval motifs may also play a role in the alignment of GPCR oligomers, because many of these motifs are found within the primary sequences of several GPCRs.
3.1. Y-Aminobutyric Acid BR1-y-Aminobutyric Acid BR2 Hetero-Oligomerization
The Y-aminobutyric acid B (GABAb) receptors provide a significant example of the importance of GPCR oligomerization in receptor trafficking, as illustrated by the interaction between GABABR1 and GABABR2, which share a 35% overall amino acid homology, that results in the formation of a fully functional GABAB receptor (60-64). When individually expressed, GABABR1 is mostly intracellularly localized within the ER (65), whereas the GABABR2 is expressed at the cell surface and within the cytoplasm
(62.66). Co-expression of both receptors in heterologous cell lines forms GABABR1-GABABR2 hetero-oligomers, which display functional characteristics similar to endogenous GABAB receptors (60-62,64), and increases GABABR1 cell-surface expression (61). Interactions between the coiled-coil domains within the carboxyl termini of the GABABR1 and GABABR2
(60.67) and their transmembrane domains (68,69) participate in hetero-oli-gomer formation (60-64). The coiled-coil domain also masks the ER retention motif RXR on the carboxyl terminus of GABABR1, allowing the receptor to exit the ER to be further processed within the Golgi into a mature, glycosylated receptor before cell-surface localization (61-63). The resulting glycosylation of the GABABR1 becomes resistant to the activity of the enzyme endoglycosidase H (Endo H), which specifically cleaves glycoproteins that have high-mannose oligosaccharides attached at their N-linked glycosylation sites—a characteristic of glycoproteins that are intracellularly retained in the ER (61-63).
3.2. Hetero-Oligomerization Between <x1-AR Subtypes
A further example demonstrating the importance of GPCR oligomerization in receptor cell-surface trafficking is the interaction of the a1B-AR with the a1A- or a1D-ARs, forming a1A-a1B and a1B-a1D hetero-oligomers, respectively (70). Expressed alone, a1A-AR and a1D-AR (71,72) are poorly expressed at the cell surface, whereas the a1B-AR is predominantly expressed at the cell surface (73). The formation of hetero-oligomers resulted in no change in the pharmacology of either receptor but increased cell-surface expression of the a1A- and a1D-ARs, compared to the a1A- and a1D-ARs expressed alone (70). These observations suggest that the a1B-AR facilitates transport of a1A- and a1D-ARs to the cell surface (70). Physical interactions forming hetero-oligomers are not mediated by interactions between the carboxyl or amino terminus of the receptors and are specific, because co-expression of a1A- and a1D-ARs did not lead to formation of hetero-oligomers or change in cell-surface expression of either receptor.
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