G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the ␣-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that ␣-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired ␣-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes ␣-factor receptor signal transduction. Structurefunction studies suggested that the GXXXG motif in TM1 of the ␣-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.
Age-related disease, not aging per se, causes most morbidity in older humans. Here we report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in three mouse models. In a longevity study, median survival was increased in UCP mice (animals with skeletal muscle-specific UCP1 expression), and lymphoma was detected less frequently in UCP female mice. In apoE null mice, a vascular disease model, diet-induced atherosclerosis was decreased in UCP animals. In agouti yellow mice, a genetic obesity model, diabetes and hypertension were reversed by induction of UCP1 in skeletal muscle. Uncoupled mice had decreased adiposity, increased temperature and metabolic rate, elevated muscle SIRT and AMP kinase, and serum characterized by increased adiponectin and decreased IGF-1 and fibrinogen. Accelerating metabolism in skeletal muscle does not appear to impact aging but may delay age-related disease.
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.
The ␥ subunits of heterotrimeric G proteins are required for receptor-G protein coupling. The C-terminal domains of G␥ subunits can contact receptors and influence the efficiency of receptor-G protein coupling in vitro. However, it is unknown whether receptor interaction with the C terminus of G␥ is required for signaling in vivo. To address this question, we cloned G␥ homologs with diverged C-terminal sequences from five species of budding yeast. Each G␥ homolog functionally replaced the G␥ subunit of the yeast Saccharomyces cerevisiae (STE18 gene product). Mutagenesis of S. cerevisiae Ste18 likewise indicated that specific C-terminal sequence motifs are not required for signaling. Strikingly, an internal in-frame deletion removing sequences preceding the C-terminal CAAX box of Ste18 did not impair signaling by either of its cognate G protein-coupled pheromone receptors. Therefore, receptor interaction with the C-terminal domain of yeast G␥ is not required for receptor-mediated G protein activation in vivo. Because the mechanism of G protein activation by receptors is conserved from yeast to humans, mammalian receptors may not require interaction with the tail of G␥ for G protein activation in vivo. However, receptor-G␥ interaction may modulate the efficiency of receptor-G protein coupling or promote activation of G␥ effectors that co-cluster with receptors.
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