Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase.

Hingorani, Vijay N.; Tobias, D T; Henderson, Jeffrey T.; Ho, Yee-Kin

doi:10.1016/s0021-9258(18)68731-9

Cited by 69 publications

(14 citation statements)

References 49 publications

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“…The activity of PDE from which only one of the two regulatory subunits has been removed by trypsin digestion was previously reported to be 50% of the activity of PDE with no 7 (Deterre et al, 1988). The discrepancy with our results again suggests that trypsin-activated PDE is different from GGTP-activated PDE (Navon & Fung, 1987;Hingorani et al, 1988;Sitaramayya et al, 1986); if the value of is indeed higher when PDE is activated by GGTP7S instead of Ggxp, one of the possible explanations could therefore be a lesser stability of the G-PDE complex when GTP is replaced by GTP7S, leading to increased dissociation of Ga0XP-P7 from Pa(j.…”

Section: Discussioncontrasting

confidence: 95%

“…Incubation of the PDE with trypsin degrades the P7 subunit and at the same time activates the PDE, while addition of P7 to trypsin-activated PDE inhibits the enzyme (Hurley & Stryer, 1982): by analogy, it has been proposed that light-induced activation of PDE is achieved by removal of the inhibitory subunit from the Pa(3 subunits by GaGTP (Yamazaki et al, 1983); a complex of P7 and G^jp has indeed been isolated (Deterre et al, 1986). The GaGTP-Pa)37 complex is however stable enough to be precipitated with anti-G" monoclonal antibody (Navon & Fung, 1987) and to give rise to cross-linked products by GaGXP or G^a nd Pa/3 (Hingorani et al, 1988). Kinetic evidence moreover suggests that the 1 Abbreviations: PDE, cGMP phosphodiesterase; P7, inhibitory subunit of PDE; Pap, catalytic subunits of PDE; R*, excited rhodopsin; G, GTP-binding protein or G-protein; G" and GSy, subunits of G; G^jp, a subunit of G with GTP bound.…”

mentioning

confidence: 99%

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Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

Bennett¹,

Clerc²

1989

Biochemistry

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The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G alpha GTP (alpha subunit of the G-protein with GTP bound) with high affinity (100 +/- 50 nM) partially activates the enzyme (Vmax1 approxmately 0.05 Vmax to 0.10V max to trypsin-activated phosphodiesterase); binding of a second G alpha GTP with lower affinity (600 +/- 100 nM) induces maximal activation (Vmax2 approximately Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP-gamma S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.

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Section: Discussioncontrasting

confidence: 95%

mentioning

confidence: 99%

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

Bennett¹,

Clerc²

1989

Biochemistry

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“…The physical state of the αt.GDP in the membrane may play an important role in the interaction with PDE. The finding that the α-subunit of transducin forms dimers and perhaps trimers [49,56,57] supports the hypothesis that G-proteins may function as oligomers [34,58,59]. The crystal structure of G i α1 [60] shows that the amino terminus of the α-subunit of G i forms extensive quaternary contacts with neighboring α-subunits in the lattice in a head to tail arrangement, strongly supporting the multimers hypothesis of α-subunits or heterotrimers.…”

Section: Discussionsupporting

confidence: 58%

Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin α-subunit

1999

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In the present report, the region of interaction between the GDP-bound alpha-subunit of transducin (alphat.GTP) and the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) has been studied. It is widely accepted that the alphat.GTP is the active form of transducin and that the GDP-bound transducin alpha-subunit (alphat. GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pgamma on alphat.GTP is in a region between the exposed face of the alpha3 and alpha4 helices of alphat.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900-26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP)-derivatized Pgamma (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pgamma to alphat.GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound transducin alpha-subunit (alphat-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68-->Ala Pgamma mutant, and by pretreatment of the alphat.GDP with the betagamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pgamma specifically transferred the [125I]APC group to Cys-250 and Cys-210 of alphat.GDP. These data indicate that the C-terminal region (especially Cys-68-Trp-70) of Pgamma interacts with alphat. GDP on the exposed interface between alpha2/beta4 and alpha3/beta5 of the alpha-subunit of transducin. Disulphide exchange was also observed with the alpha-subunit of holotransducin but this was only approx. 60% of that of pure alphat.GDP. The variation in the binding pattern between alphat.GDP and alphat.GTP with the C-terminal region of Pgamma may contribute to the functional difference between the GDP- and GTP-bound states.

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“…(/) The carboxyl-terminal region of ax is involved in the interaction with Byt Recently, it has been suggested on the basis of cross-linking studies that the 5-kDa COOH terminus of at directly interacts with the amino-terminal region of the Byx subunit (Hingorani et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

“…Hingorani et al (1988) studying the chemically cross-linked products of Gt subunits, have shown that the 5-kDa COOH-h i b i t e d .…”

mentioning

confidence: 99%

Effect of monoclonal antibody binding on .alpha.-.beta..gamma. subunit interactions in the rod outer segment G protein, Gt

Mazzoni

Hamm²

1989

Biochemistry

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The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

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Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase.

Cited by 69 publications

References 49 publications

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin α-subunit

Effect of monoclonal antibody binding on .alpha.-.beta..gamma. subunit interactions in the rod outer segment G protein, Gt

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