Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

Abstract

The concept that G protein-coupled receptors (GPCRs) 1 exist as dimers or higher order oligomers has moved rapidly from hypothesis to being widely accepted (1-4). A range of approaches has contributed to this understanding. This includes the ability to co-immunoprecipitate differentially epitopetagged forms of a GPCR from cells in which they are co-expressed, and, in intact cells, the application of a number of resonance energy transfer-based techniques. However, the role of dimerization in function and the mechanisms then responsible for initiation of signal transduction by the dimer have been more recalcitrant to analysis. Significant progress in this area has recently been achieved for the class C GPCRs. These contain both a long extracellular N-terminal domain, to which agonist ligands bind, and the prototypic seven transmembrane (TM) helix bundle architecture that is the common feature of all GPCR families (for review, see Ref. 5). The functional ␥-aminobutyric acid, type b receptor is a hetero-dimer of two distinct gene products in which trafficking to the plasma membrane requires interaction between the partner polypeptides (6 -9). This indicates that a key role of dimerization is achieving appropriate cellular localization. This is also true for the class A rhodopsin-like GPCRs because non-functional, truncated splice variants can restrict plasma membrane delivery of fulllength GPCRs and, thus, limit their function (10 -12). Chimeric class C GPCRs consisting of the extracellular domain of one GPCR and the TM and intracellular elements of a second, closely related GPCR or in which the intracellular loops of dimer partners are exchanged have provided strong evidence that the mechanism of action of the dimer involves transactivation (13)(14); that is, ligand binding to one element of the dimer results in activation of G protein produced by the other GPCR within the dimer. Again, the ␥-aminobutyric acid, type b receptor has been particularly informative in this regard as only one of the two gene products that forms the dimer is able to bind the agonist ␥-aminobutyric acid. Equivalent systems are not available for the majority of class A GPCRs. However, for the luteinizing hormone receptor, which also has a long N-terminal domain that binds the ligand, partial reconstitution of function has been achieved by co-expression of distinct pairs of mutants (15-16). Furthermore, in recent studies co-expression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescued hormone-activated cAMP production (17).

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

“…For example, agonist binding to GPCRs such as cardiac muscarinic acetylcholine receptors is cooperative (21,22). Furthermore, a recent investigation using ␣1b-adrenergic or histamine H1 receptors fused at their C termini with G␣ 11 has shown that G 11 activation occurs upon co-expression of one fusion consisting of wild type G 11 and a receptor defective in G protein activation with a second fusion protein consisting of a wild type receptor and mutant G 11 that cannot release GDP (54). This study also showed that a fusion consisting of TM1 of the ␣1b-adrenergic receptor and G 11 could associate with wild type ␣1b-adrenergic receptors but that agonist stimulation did not activate the TM1-G 11 fusion.…”

Visual Rhodopsin Sees the Light: Structure and Mechanism of G Protein Signaling

“…18 and 19). Experimental evidence to support such a model comes from studies showing that the G t -activating potential of detergentsolubilized rhodopsin dimers exceeds that of monomer preparations (45) and trans-activation studies with other Family A GPCRs that employ fusion proteins between active and inactive receptors and G protein ␣-subunit (G ␣ ) subunits (46). The isolation of a pentameric complex between a dimeric leukotriene B4 receptor BLT1 and the G protein heterotrimer is also consistent with this view (47).…”

Functional Homomers and Heteromers of Dopamine D2L and D3 Receptors Co-exist at the Cell Surface

“…G protein coupling of D 2 receptors has been well characterized with a general consensus for promiscuous coupling to G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o1 (29,33,57). By contrast, the G protein coupling profile of D 3 receptors has proven more challenging to define: they appear to be most efficient in coupling to G␣ o Table 4 for quantitative details.…”

“…For the second set, mutations were introduced into the receptors such that a pair of hydrophobic residues located at equivalent positions in the 2nd intracellular loop of each of the D 2L and D 3 receptors was converted to acidic residues. As shown previously this provides a generic means in the rhodopsin family G protein-coupled receptors to eliminate G protein activation in response to agonists without alteration of the ligand binding pocket (33). These changes produced V136E,M140D-D 2L and V132E,V136D-D 3 receptors and each of these was then linked to C351I-G␣ o .…”

“…To do so we employed the potential for functional complementation following co-expression of pairs of nonequivalent and nonfunctional fusions between dopamine receptors and a G protein they are usually able to activate. Although the D 2L receptor interacts effectively with each member of the pertussis toxinsensitive G␣ i family of G proteins (29), the D 3 receptor appears to be more selective, showing greatest activation of G␣ o (30,33). We therefore generated fusion proteins containing either the D 2L or D 3 receptors linked to a pertussis toxin-insensitive, C351I, a variant of G␣ o that both receptors still activate (29,30).…”