How a G Protein Binds a Membrane

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments However, there are no reports of a nonmammalian species having two distinct transducins In the present study, we report the cloning and cellular localization of two transducin a subunits (G alpha t) from the tiger salamander Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins The salamander G alpha t-1 has 912-937% amino acid sequence identity to mammalian rod G alpha t subunits and 797-809% to mammalian cone Gats The salamander G alpha t-2 has 862-879% sequence identity to mammalian cone G alpha ts and 789-809% to mammalian rod G alpha ts at the amino acid level The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes Northern blot analysis identified retina-specific transcripts of 30 kb for rod G alpha t and 26 kb for cone G alpha t Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors

Salamander rods and cones contain distinct transducin alpha subunits.

Previously, we have demonstrated that somatostatin mediates all of its inhibitory effects on glucose-induced insulin secretion from the HIT-T15 cell through pertussis toxin-sensitive G-proteins and that the membrane fraction of this clonal line of pancreatic beta-cells contains six such proteins: G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and three forms of G(o) alpha. To determine the specificity of somatostatin receptor-G-protein coupling in HIT-T15 cells, we examined the ability of antisera specific for the COOH-terminus of G alpha subtypes to inhibit somatostatin-induced augmentation of membrane GTPase activity. GTPase activity increased in membranes as a function of GTP. At all concentrations of GTP studied, 1 mumol/l somatostatin stimulated GTPase activity. Pertussis-toxin pretreatment prevented the effects of somatostatin. Antisera selective for G(o) alpha subtypes reduced the effects of somatostatin on GTPase activity (GTPase activity in absence of antisera, 125 +/- 3% of control; in the presence of antisera 976, 110 +/- 2% of control; n = 13, P < 0.001), whereas antisera directed against G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and Gs alpha were without effect. Somatostatin also significantly prevented cyclic AMP accumulation during perifusion with 11.1 mmol/l glucose through a pertussis toxin-sensitive mechanism. These data indicate that the somatostatin receptor couples to G(o) alpha in the HIT-T15 cell and suggest that G(o) alpha may link somatostatin to cyclic AMP metabolism in pancreatic beta-cells.

Somatostatin selectively couples to G(o) alpha in HIT-T15 cells.

The molecular basis of the interaction between the visual receptor, rhodopsin, and the rod outer segment GTP-binding protein, transducin or Gt, was studied using a synthetic-peptide-competition approach to elucidate the site(s) on the Gt alpha-subunit (alpha t) involved in high-affinity binding to light-activated rhodopsin (R*). Synthetic peptides based on the amino acid sequence of portions of the molecule that interact with rhodopsin can themselves bind the rhodopsin and thus behave as competitive inhibitors of rhodopsin-G-protein interaction. This blockade was assessed by measuring the ability of peptides to inhibit Gt stabilization of the metarhodopsin II conformation of rhodopsin. Based upon this analysis, two regions near the C-terminal of alpha 1 are important for interaction with R*.

The molecular basis of GTP-binding protein interaction with receptors.

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The molecular basis of GTP-binding protein interaction with receptors.