An antibody directed against the carboxyl-terminal decapeptide of the α subunit of the retinal GTP-binding protein, transducin. Effects on transducin function

R. A. Cerione, S. Kroll, R. Rajaram, C. Unson, P. Goldsmith, Allen M. Spiegel

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the α subunit of transducin (α(T)) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified α(T) and β/γ (βγ(T)) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or α(T)) to AS/7 ≃ 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the α(T) subunit and the βγ(T) complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free α(T), holo-transducin, or α(T) in the presence of excess βγ(T), prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of α(T) to bind or hydrolyze GTP and, in fact, the incubation of α(T) with AS/7 results in a stimulation of the intrinsic GTPase activity for α(T) alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-α(T) interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated α(T) subunit. Specifically, when the pure α(T)-guanosine 5-O-(3-thiotriphosphate) (α(T)GTPγS) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with α(T)GTPγS, alone, or with α(T)GTPγS preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of α(T); the inactive α(T)GDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the α(T) subunit highlight the existence of distinct functional domains on α(T). In addition these results suggest that the interactions (by the photoreceptor) at the carboxyl-terminal domain of α(T) are not only essential for rhodopsin-transducin coupling but may also serve to facilitate the effective coupling of an activated α(T) subunit to the effector enzyme, PDE.

Original languageEnglish (US)
Pages (from-to)9345-9352
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number19
StatePublished - 1988
Externally publishedYes

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Transducin
Rhodopsin
GTP-Binding Proteins
Antibodies
GTP Phosphohydrolases
Phosphoric Diester Hydrolases
Cyclic GMP
Assays
Guanosine 5'-O-(3-Thiotriphosphate)
Blocking Antibodies
Guanosine Triphosphate
Hydrolysis
Phospholipids
Immunoglobulin G
Rabbits
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

An antibody directed against the carboxyl-terminal decapeptide of the α subunit of the retinal GTP-binding protein, transducin. Effects on transducin function. / Cerione, R. A.; Kroll, S.; Rajaram, R.; Unson, C.; Goldsmith, P.; Spiegel, Allen M.

In: Journal of Biological Chemistry, Vol. 263, No. 19, 1988, p. 9345-9352.

Research output: Contribution to journalArticle

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abstract = "An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the α subunit of transducin (α(T)) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified α(T) and β/γ (βγ(T)) subunit components, with the highest levels of inhibition (80-95{\%}) occurring under conditions where the molar ratio of holo-transducin (or α(T)) to AS/7 ≃ 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the α(T) subunit and the βγ(T) complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free α(T), holo-transducin, or α(T) in the presence of excess βγ(T), prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of α(T) to bind or hydrolyze GTP and, in fact, the incubation of α(T) with AS/7 results in a stimulation of the intrinsic GTPase activity for α(T) alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-α(T) interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated α(T) subunit. Specifically, when the pure α(T)-guanosine 5-O-(3-thiotriphosphate) (α(T)GTPγS) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with α(T)GTPγS, alone, or with α(T)GTPγS preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of α(T); the inactive α(T)GDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the α(T) subunit highlight the existence of distinct functional domains on α(T). In addition these results suggest that the interactions (by the photoreceptor) at the carboxyl-terminal domain of α(T) are not only essential for rhodopsin-transducin coupling but may also serve to facilitate the effective coupling of an activated α(T) subunit to the effector enzyme, PDE.",
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T1 - An antibody directed against the carboxyl-terminal decapeptide of the α subunit of the retinal GTP-binding protein, transducin. Effects on transducin function

AU - Cerione, R. A.

AU - Kroll, S.

AU - Rajaram, R.

AU - Unson, C.

AU - Goldsmith, P.

AU - Spiegel, Allen M.

PY - 1988

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N2 - An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the α subunit of transducin (α(T)) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified α(T) and β/γ (βγ(T)) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or α(T)) to AS/7 ≃ 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the α(T) subunit and the βγ(T) complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free α(T), holo-transducin, or α(T) in the presence of excess βγ(T), prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of α(T) to bind or hydrolyze GTP and, in fact, the incubation of α(T) with AS/7 results in a stimulation of the intrinsic GTPase activity for α(T) alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-α(T) interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated α(T) subunit. Specifically, when the pure α(T)-guanosine 5-O-(3-thiotriphosphate) (α(T)GTPγS) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with α(T)GTPγS, alone, or with α(T)GTPγS preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of α(T); the inactive α(T)GDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the α(T) subunit highlight the existence of distinct functional domains on α(T). In addition these results suggest that the interactions (by the photoreceptor) at the carboxyl-terminal domain of α(T) are not only essential for rhodopsin-transducin coupling but may also serve to facilitate the effective coupling of an activated α(T) subunit to the effector enzyme, PDE.

AB - An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the α subunit of transducin (α(T)) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified α(T) and β/γ (βγ(T)) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or α(T)) to AS/7 ≃ 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the α(T) subunit and the βγ(T) complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free α(T), holo-transducin, or α(T) in the presence of excess βγ(T), prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of α(T) to bind or hydrolyze GTP and, in fact, the incubation of α(T) with AS/7 results in a stimulation of the intrinsic GTPase activity for α(T) alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-α(T) interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated α(T) subunit. Specifically, when the pure α(T)-guanosine 5-O-(3-thiotriphosphate) (α(T)GTPγS) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with α(T)GTPγS, alone, or with α(T)GTPγS preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of α(T); the inactive α(T)GDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the α(T) subunit highlight the existence of distinct functional domains on α(T). In addition these results suggest that the interactions (by the photoreceptor) at the carboxyl-terminal domain of α(T) are not only essential for rhodopsin-transducin coupling but may also serve to facilitate the effective coupling of an activated α(T) subunit to the effector enzyme, PDE.

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