Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems (original) (raw)
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The Journal of biological chemistry, 1986
The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neithe...
Amino Acid Sequence of the alpha Subunit of Transducin Deduced from the cDNA Sequence
Proceedings of The National Academy of Sciences, 1985
Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes G, and G1, the regulatory proteins of adenylate cyclase. Here we Abbreviations: Ta, a subunit of transducin; T /3y, -'y subunit complex of transducin; PDEase, phosphodiesterase, p[NH]ppG, guanosine 5'-[A3,y-imido]triphosphate; R*, photolyzed rhodopsin; G, and Gi, stimulatory and inhibitory coupling proteins of hormone-sensitive adenylate cyclase. cPresent address:
The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (G t ) by binding to the ␥ subunits of G t and preventing their association with the G t ␣ subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits G t subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of G t s available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca 2؉ and calmodulin. The Ca 2؉ dependence was within the physiological range of Ca 2؉ concentrations in rods (K1 ͞2 ؍ 230 ؎ 9 nM) and was highly cooperative (n app ؍ 3.6 ؎ 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca 2؉ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca 2؉ (8 nM). No evidence for Pd phosphorylation by other Ca 2؉ -dependent kinases was found. These results suggest that Ca 2؉ can regulate the light response at the level of G t activation through its effect on the phosphorylation state of Pd.
Biochemical Journal, 2009
Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Gαt (the transducin α subunit) and retGC. Gαt co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC–Gαt complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both Gαt and retGC. The Gαt–retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Gαt stronger than the GTP[S] (GTPγS; guanosine 5′-[γ-thio]triphos...
Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin
The EMBO journal, 1985
Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T al...
Improved purification of transducin subunits from bovine retinal rod outer segments
1995
Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin.
2007
PURPOSE. Rhodopsin mutations account for approximately 25% of human autosomal dominant retinal degenerations. However, the molecular mechanisms by which rhodopsin mutations cause photoreceptor cell death are unclear. Mutations in genes involved in the termination of rhodopsin signaling activity have been shown to cause degeneration by persistent activation of the phototransduction cascade. This study examined whether three disease-associated rhodopsin substitutions Pro347Ser, Lys296Glu, and the triple mutant Val20Gly, Pro23His, Pro27Leu (VPP) caused degeneration by persistent transducinmediated signaling activity. METHODS. Transgenic mice expressing each of the rhodopsin mutants were crossed onto a transducin ␣-subunit null (Tr ␣ Ϫ/Ϫ ) background, and the rates of photoreceptor degeneration were compared with those of transgenic mice on a wild-type background. RESULTS. Mice expressing VPP-substituted rhodopsin had the same severity of degeneration in the presence or absence of Tr ␣ . Unexpectedly, mice expressing Pro347Ser-or Lys296Glusubstituted rhodopsins exhibited faster degeneration on a Tr ␣ Ϫ/Ϫ background. To test whether the absence of ␣-transducin contributed to degeneration by favoring the formation of stable rhodopsin/arrestin complexes, mutant Pro347Ser ϩ , Tr ␣ Ϫ/Ϫ mice lacking arrestin (Arr Ϫ/Ϫ ) were analyzed. Rhodop-sin/arrestin complexes were found not to contribute to degeneration. CONCLUSIONS. The authors hypothesized that the decay of metarhodopsin to apo-opsin and free all-trans-retinaldehyde is faster with Pro347Ser-substituted rhodopsin than it is with wild-type rhodopsin. Consistent with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H 2 , which form from retinaldehyde, were elevated in Pro347Ser transgenic mice. (Invest Ophthalmol Vis Sci. 2007;48:5445-5453)
Journal of Biological Chemistry
We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the az-adrenergic receptor (azAR) which has been partially purified (-500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and N., bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the a2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the a-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the azAR and Ni has been established. The maximum turnover number for the azAR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the &adrenergic receptormediated isoproterenol-stimulated GTPase activity in N, and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein).
Regulation of the Kinetics of Phosducin Phosphorylation in Retinal Rods.
Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-protein (G) signaling. Unphosphorylated Pd binds to G␥ subunits and blocks their interaction with G␣. This binding sequesters G␥ and inhibits both receptor-mediated activation of G␣ and direct interactions between G␥ and effector enzymes. When phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of G␥. To further understand the role of Pd in regulating G-protein signaling in retinal rod photoreceptor cells, we have measured the abundance of Pd in rods and examined factors that control the rate of Pd phosphorylation. Pd is expressed at a copy number comparable to that for the rod G-protein, transducin (G t ). The ratio of rhodopsin (Rho) to Pd is 15.5 ؎ 3.5 to 1. The rate of Pd phosphorylation in rod outer segment preparations was dependent on [cAMP]. K1 ⁄2 for cAMP was 0.56 ؎ 0.09 M, and the maximal rate of phosphorylation was ϳ500 pmol PO 4 incorporated/min/ nmol Rho. In the presence of G t ␥ this rate was decreased ϳ50-fold. From these data, one can estimate a t1 ⁄2 of ϳ3 min for the rephosphorylation of Pd in rods during the recovery period after a light response. This relatively slow rephosphorylation of the Pd⅐G t ␥ complex may provide a period of molecular memory in which sensitivity to further light stimuli is reduced as a result of sequestration of G t ␥ by Pd.