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

Abstract

G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast ␣-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding-and G protein coupling-defective ␣-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding-and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the ␣-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.

G Protein Activation by the Leukotriene B4 Receptor Dimer

“…For instance, co-expression of ligand binding-defective and G protein coupling-defective mutant ␣-factor receptors did not significantly improve signaling, suggesting that the signal of agonist binding was not transferred from the G protein coupling-defective mutant to the agonist-binding defective mutant (38). Co-expression of different mutants of the angiotensin II receptor could not restore signaling (39), indicating that complete trans-complementation did not occur in this case either.…”

Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

“…Consistent with this view, Ldb19 bound less avidly than did either Rod1 or Rog3 to the isolated Ste2 tail in vitro, presumably because Ldb19 recognizes an additional determinant only accessible in a misfolded receptor. For example, because native Ste2 functions as a dimer (125)(126)(127), perhaps Ldb19 associates with the tail and also a site exposed in a monomer when the dimer dissociates. In any event, a role in removal of misfolded molecules may explain why overexpression of Ldb19 was unable to enhance the rate of recovery in the adaptation assay, which is conducted under conditions where the properly folded state of the receptor is stabilized by ligand binding.…”

Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors

“…(The number of ␣-factor receptors has been reported to approximately equal the number of trimeric G proteins in cells containing normal chromosomal copies of the genes encoding receptors and G protein subunits [20,56].) However, oligomerization of ␣-factor receptors has now been demonstrated based on fluorescence (10,40) and bioluminescence (17) energy transfer, coimmunoprecipitation of differentially tagged receptors (55), and cotrafficking of different receptor alleles (55), raising the possibility that functional interactions might, instead, be mediated by biochemical interactions among co-oligomerized receptors. Interpretation of experiments involving overexpression of G protein subunits is complicated by the fact that subtle differences in the relative amounts of the negative regulatory G protein ␣ subunits with respect to positive regulatory ␤ and ␥ subunits can lead to either increased or decreased responses to ␣-factor (4, 11).…”

“…However, interpretation of these results is complicated by the fact that the loss of dominant effects upon overexpression of G protein was not complete (28) and by the concerns that any imbalance in the expression levels of the three separately encoded G protein subunits can affect activation of the pheromone response pathway (4,11). The second type of explanation for functional interactions between receptors is based on the extensive evidence for physical interactions between co-oligomerized receptors (10,17,39,55). If signaling requires functional interactions between receptors within an oligomer, co-oligomerization of a normal receptor with a defective mutant receptor could be sufficient to inhibit signaling by the normal receptor and co-oligomerization of a normal receptor with constitutive or hypersensitive mutants could suppress abnormal responses by the mutant receptors.…”