Emir Duzic

Receptor-independent Activators of Heterotrimeric G-protein Signaling Pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-proteinsignaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. β-galactosidase reporter assays in yeast strains expressing different Gα subunits (Gpa1, Gsα, Giα2 (Gpa1(1–41)), Giα3(Gpa1(1–41)), Gα16(Gpa1(1–41))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the Giα2 and Giα3genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gβγ, whereas AGS3 bound Gα and exhibited a preference for GαGDP versus GαGTPγS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Gα and Gβγ that allow the subunits to subserve functions that do not require initial heterotrimer formation.

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Gα hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Gα. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration.

Utilizing a functional screen in the yeastSaccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-proteinsignaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Gαi/Gαo heterotrimeric signaling pathways. In addition, the in vivo and in vitroproperties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Gαi/Gαo. AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.

The 3′-Untranslated Region of the α2C-Adrenergic Receptor mRNA Impedes Translation of the Receptor Message

We report that two subtypes of α2-adrenergic receptors (α2A/D- and α2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3′-untranslated region (3′-UTR) of the α2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, α2C-AR; coding region plus 1140 nt, α2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 α2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 α2A/D-AR transfectants expressed receptor protein (200–5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3′-UTR of the α2C-AR in mRNA processing was determined by generating new constructs in which the 3′-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3′ to the stop codon. Truncation of the 3′-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The α2C-AR mRNA with the longer 3′-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating α2C-AR expression and associated signaling events.

Evaluation of agonist efficacy and receptor reserve for alpha 2D-adrenergic receptor regulated G protein activation in PC12 cell membranes.

The receptor-coupling efficiency for epinephrine (EPI) stimulated heterotrimeric G protein activation was studied at the G protein level in membranes prepared from PC12 cells expressing cloned alpha 2D-adrenergic receptors (alpha 2D-AR). After pretreatment with different concentrations of N-ethoxycarbonyl-1,2-dihydroquinoline, which irreversibly inactivates alpha 2D-AR, the portion of alpha 2D-ARs remaining active (q) was estimated from EPI-stimulated [35S]GTP gamma S binding. This function-derived estimate was close to the actual remaining number of receptors, as determined in saturation-binding studies using the selective alpha 2-AR antagonist [3H]rauwolscine in the same membranes. The agonist dissociation constant (KA) derived from EPI-stimulated [35S]GTP gamma S binding via Furchgott analysis was similar to the EC50 of EPI in the same assay, but 40-fold lower than its Ki measured from EPI competition for [3H]rauwolscine-binding sites in the presence of GTP gamma S and Na+. The occupancy-response relationship, calculated using Ki rather than KA, was markedly nonlinear, consistent with the high expression of alpha 2D-AR in these membranes. A nonlinear occupancy-response relationship was more directly confirmed by measuring the maximal level (i.e., full occupancy level) of G protein activation at graded densities of alpha AD-AR after N-ethoxycarbonyl-1,2-dihydroquinoline treatment. Determination of the number of G-proteins activated per receptor yielded lower values at higher receptor densities, indicating that overexpression of receptors can reduce their efficiency. Our results indicate the potential utility of using GTP-binding studies to assess agonist efficacy at the G protein level under conditions where receptor occupation can also be directly measured.