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PubMed2002

Mapping of a novel locus for achromatopsia (ACHM4) to 1p and identification of a germline mutation in the alpha subunit of cone transducin (GNAT2).

OBJECTIVE: To determine the molecular basis for achromatopsia using autozygosity mapping and positional candidate gene analysis. DESIGN AND METHODS: A large consanguineous Pakistani family containing six subjects with autosomal recessive complete achromatopsia was ascertained. After excluding linkage to the two known achromatopsia genes (CNGA3 and CNGB3), a genome wide linkage screen was undertaken. RESULTS: Significant linkage was detected to a 12 cM autozygous segment between markers D1S485 and D1S2881 on chromosome 1p13. Direct sequence analysis of the candidate gene GNAT2 located within this interval identified a frameshift mutation in exon 7 (c842_843insTCAG; M280fsX291) that segregated with the disease. CONCLUSIONS: The GNAT2 gene codes for cone alpha-transducin, the G protein that couples the cone pigments to cGMP-phosphodiesterase in phototransduction. Although cone alpha-transducin has a fundamental role in cone phototransduction, mutations in GNAT2 have not been described previously. Since mutations in the CNGA3 gene may cause a variety of retinal dystrophies (complete and incomplete achromatopsia and progressive cone dystrophy), GNAT2 mutations may also prove to be implicated in other forms of retinal dystrophy with cone dysfunction.

Amino Acid Sequence↗
PubMed2003

Cone photoreceptor betagamma-transducin: posttranslational modification and interaction with phosducin.

PURPOSE: To characterize the structure of cone betagamma-transducin (Tbeta3gamma8) and its interaction with phosducin (pdc). METHODS: The Tgamma8 subunit of Tbeta3gamma8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tbeta3gamma8 was compared with rod betagamma-transducin (Tbeta1gamma1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tbeta1gamma1 was determined. RESULTS: The mature Tgamma8 is a 65-amino-acid peptide encoded by the Ggamma8 gene with an acetylated and a farnesylated-methylated N- and C-terminus, respectively. Purified Tbeta3gamma8 is similar to Tbeta1gamma1 in that (1) both are heterogeneous, containing methylated and demethylated Tgamma subunits; (2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tgamma. Tbeta3gamma8 differs from Tbeta1gamma1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tgamma methylation. CONCLUSIONS: Tbeta3gamma8 is identical with Tbeta1gamma1 in Tgamma isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tbetagamma characteristics by Tgamma methylation reveal conformational changes on a surface domain that is essential for Tbetagamma functions and support a regulatory role for reversible methylation.

Amino Acid Sequence↗
PubMed2005

Purification, reconstitution on lipid vesicles, and assays of PDE6 and its activator G protein, transducin.

PDE6 in rod and cone photoreceptors is the principal effector of phototransduction. It is kept at a very low activity level in the dark, and in the light it is strongly activated by the guanosine 5'-triphosphate-bound form of the alpha-subunit of the G protein, transducin. Both transducin and PDE6 are peripheral proteins, and understanding both their interactions with one another and the roles of lipids in their function requires reconstituting purified proteins on the surfaces of defined lipid bilayers. We describe here methods for purifying the proteins, reconstituting them with vesicles, and assaying catalytic activity and binding.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Umami taste responses are mediated by alpha-transducin and alpha-gustducin.

The sense of taste comprises at least five distinct qualities: sweet, bitter, sour, salty, and umami, the taste of glutamate. For bitter, sweet, and umami compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors in specialized epithelial cells located in the taste buds, leading to the activation of signal transduction cascades. Alpha-gustducin, a taste cell-expressed G-protein alpha subunit closely related to the alpha-transducins, is a key mediator of sweet and bitter tastes. Alpha-gustducin knock-out (KO) mice have greatly diminished, but not entirely abolished, responses to many bitter and sweet compounds. We set out to determine whether alpha-gustducin also mediates umami taste and whether rod alpha-transducin (alpha(t-rod)), which is also expressed in taste receptor cells, plays a role in any of the taste responses that remain in alpha-gustducin KO mice. Behavioral tests and taste nerve recordings of single and double KO mice lacking alpha-gustducin and/or alpha(t-rod) confirmed the involvement of alpha-gustducin in bitter (quinine and denatonium) and sweet (sucrose and SC45647) taste and demonstrated the involvement of alpha-gustducin in umami [monosodium glutamate (MSG), monopotassium glutamate (MPG), and inosine monophosphate (IMP)] taste as well. We found that alpha(t-rod) played no role in taste responses to the salty, bitter, and sweet compounds tested or to IMP but was involved in the umami taste of MSG and MPG. Umami detection involving alpha-gustducin and alpha(t-rod) occurs in anteriorly placed taste buds, however taste cells at the back of the tongue respond to umami compounds independently of these two G-protein subunits.

Animals↗
PubMed1996

The differences in the expressions of visual pigments and transducin in photoreceptor cell differentiation.

The distribution and accumulation of visual pigments, i.e., rod pigment, rhodopsin and red sensitive cone pigment, iodopsin, and transducin in the retina of chicken and chicken embryo were investigated immunohistochemically using their specific antibodies. The immunoreactivities of these proteins appeared at the early stage of photoreceptor differentiation (embryonal day 15) and increased in the photoreceptor cells appeared to reach maximum at the end of the embryonal period (embryonal day 20). On the other hand, although the immunoreactivity of beta gamma subunit of transducin (T beta gamma) was detected at embryonal day 15, the expression level of T beta gamma still remained in low level during the embryonal period. These observations suggest that both T beta gamma and visual pigments are expressed during the embryonic period in chicken photoreceptor cells, but their accumulations in the cells are different.

Animals↗
PubMed1991

Early disappearance of alpha-transducin in light-induced photoreceptor degeneration in albino rats.

Progressive degeneration of retinal photoreceptor cells occurs in albino rats when exposed to continuous lighting. Three proteins involved in the phototransduction cascade were immunodetected in these cells after various durations of continuous illumination. We found that S-antigen (arrestin) and rhodopsin immunoreactivities persisted for 1-2 months during the degenerative process, whilst immunoreactivity of the alpha subunit of transducin totally disappeared between day 2 and day 4 of continuous light exposure. This suggests that continuous illumination could impair alpha-transducin synthesis, a possible causal factor of photoreceptor damage.

Animals↗
PubMed1992

Stimulation and inhibition of human platelet adenylylcyclase by thiophosphorylated transducin beta gamma-subunits.

The effect of beta gamma-dimers isolated from the retinal guanine nucleotide-binding protein (G protein) transducin eluted from illuminated bovine rod outer segment membranes with GTP, guanosine 5'-O-(beta, gamma-imino)triphosphate (Gpp(NH)p), or guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) on basal and forskolin-stimulated adenylylcyclase activities in membranes of human platelets was studied. beta gamma-Subunits isolated from transducin eluted with GTP gamma S (TD beta gamma GTP gamma S) had a concentration-dependent stimulatory effect on basal adenylylcyclase activity. The stimulatory agonist prostaglandin E1 increased the potency and the maximum extent of stimulation due to TD beta gamma GTP gamma S). With a similar concentration dependence, TD beta gamma GTP gamma S exerted an inhibitory influence on forskolin-stimulated adenylylcyclase activity. At the same concentrations, beta gamma-dimers isolated with either GTP or Gpp(NH)p did not alter enzyme activities. The observed effects of TD beta gamma GTP gamma S were similar to those of directly added GTP gamma S with regard to maximum levels, time dependence, and persistence; however, TD beta gamma GTP gamma S was approximately 10-fold more potent than GTP gamma S. Treatment of TD beta gamma GTP gamma S, but not of free GTP gamma S, with hydroxylamine caused a loss of adenylylcyclase regulation by TD beta gamma GTP gamma S. The data presented indicated that TD beta gamma GTP gamma S potently and efficiently activates the stimulatory and inhibitory G proteins of adenylylcyclase in human platelet membranes. Furthermore, evidence is provided suggesting that the observed effects of TD beta gamma GTP gamma S, which can be thiophosphorylated by GTP gamma S at the beta-subunit (Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716), are due to formation of GTP gamma S at the G proteins.

Adenylyl Cyclase Inhibitors↗
PubMedDate not supplied

[Calcium-dependent interaction of transducin with calmodulin-sepharose].

It has been shown by affinity chromatography on calmodulin-sepharose that transducin, a G protein of bovine retinal rod outer segments interacts with Ca(2+)-calmodulin. This result assumes that the main part of calmodulin in dark retinal rod outer segments is associated with transducin. It has been suggested that photoactivation of retinal rods induces changes in intracellular calmodulin concentration, which may be one of the steps involved in the light adaptation of photoreceptor.

Animals↗
PubMed1989

[Inhibition of transducin by lithium: electrophysiological demonstration using the isolated retina].

Recently Avissar et al. have established that Li+ can inhibit G proteins implicated in brain function. In order to investigate the effect of Li+ on transducin, the evolution of the electroretinogram (ERG) recorded on isolated rat retina has been studied in presence of lithium. Results indicate that 10(-5) M Li+ had no effect on ERG while 10(-3) M Li+, which corresponds to therapeutic blood levels, significantly decreases ERG amplitude. This effect being nearly totally inhibited by cholera toxin (75 micrograms/l), it is concluded that Li+ acts on transducin and so inhibits the visual transduction process.

Animals↗
PubMed1989

The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin.

The functional interactions of the retinal G protein, transducin, with the cyclic GMP phosphodiesterase (PDE) have been examined using the different purified subunit components of transducin and the native and trypsin-treated forms of the effector enzyme. The limited trypsin treatment of the PDE removes the low molecular weight gamma subunit (Mr approximately 14,000) of the enzyme, yielding a catalytic moiety comprised of the two larger molecular subunits (alpha, Mr approximately 85,000-90,000; beta, Mr approximately 85,000-90,000), which is insensitive to the addition of either the pure alpha T.GTP gamma S species or the pure beta gamma T subunit complex. However, the addition of the pure alpha T.GDP species to the trypsin-treated PDE (tPDE) results in a significant (90-100%) inhibition of the enzyme activity. This inhibition can be reversed by excess beta gamma T, suggesting that the holotransducin molecule does not (functionally) interact with the tPDE. However, the inhibition by alpha T.GDP is not reversed by the alpha T.GTP gamma S complex, over a range of [alpha T.GTP gamma S] which elicits a marked stimulation of the native enzyme activity, suggesting that the activated alpha T species does not effectively bind to the tPDE. The alpha T.GDP complex also is capable of inhibiting the alpha T.GTP gamma S-stimulated cyclic GMP hydrolysis by the native PDE. This inhibition can be reversed by excess alpha T.GTP gamma S, as well as by beta gamma T, indicating that the binding site for the activated alpha T species is in close proximity and/or overlaps the binding site for the alpha T.GDP complex on the enzyme. Overall, these results are consistent with a scheme where (a) both the small and larger molecular weight subunits of PDE participate in alpha T-PDE interactions, (b) the activation of PDE by the alpha T.GTP gamma S (or alpha T.GTP) species does not result in the complete dissociation of the gamma subunit from the enzyme, and (c) the deactivation of this signal transduction system results from a direct interaction between the alpha T.GDP species and the catalytic moiety of the effector enzyme.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes.

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity.

In this work we have characterized the ability of a carboxyl peptide-specific antibody (AS/7), raised against the alpha subunit of transducin (alpha T), to potentiate the stimulation of the cyclic GMP phosphodiesterase (PDE) by transducin. The complexation of the purified guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound form of alpha T (alpha T.GTP gamma S) with AS/7 results in a 2-5-fold enhancement in the total levels of cyclic GMP hydrolysis measured after 1 min. This potentiation by AS/7 cannot be attributed simply to an increase in the apparent affinity of alpha T.GTP gamma S for the effector enzyme, nor to an increased affinity of the enzyme for the substrate cyclic GMP. The AS/7-induced potentiation is specific for alpha T.GTP gamma S-PDE interactions; this antibody has no effect on the activity of the trypsin-activated PDE nor on the ability of the GDP-bound form of alpha T to inhibit the trypsin-activated enzyme (Kroll, S., Phillips, W. J., and Cerione, R. A. (1989) J. Biol. Chem. 264, 4490-4497). Phosphatidylcholine vesicles also will enhance the alpha T.GTP gamma S-stimulated PDE activity (1.5-2-fold) relative to that measured in the absence of a lipid milieu. However, the potentiations of alpha T-stimulated cyclic GMP hydrolysis elicited by AS/7 and lipids represent separate events. Titration profiles describing the AS/7-induced potentiation, as a function of the amount of antibody added to the assay mixtures, indicate that maximal activity occurs when there is one molecule of AS/7 per two molecules of alpha T.GTP gamma S; the AS/7-induced potentiation is lost when AS/7 much greater than alpha T. GTP gamma S, i.e. conditions which favor the formation of monovalent AS/7-alpha T.GTP gamma S complexes. When the AS/7 is papain-treated to yield monovalent antibody molecules, complexation between these monovalent antibodies and alpha T still occurs (as reflected by the ability of these antibodies to block rhodopsin-alpha T coupling); however, the potentiation of the alpha T.GTP gamma S-stimulated PDE activity is lost. Taken together, these results suggest that the AS/7-induced potentiation of alpha T-stimulated activity is dependent on the bivalent nature of the antibody, and maximal stimulation of PDE activity is achieved by the interactions of two activated-alpha T molecules with a single molecule of PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Characterization of the mouse rod transducin alpha subunit gene.

A genomic clone spanning the mouse rod transducin alpha subunit (Tr alpha) gene has been isolated by screening a mouse genomic library with a bovine Tr alpha cDNA clone. The coding region of the mouse Tr alpha gene reveals an 88.7% nucleotide identify and 99.7% amino acid identity with bovine Tr alpha. The mouse Tr alpha gene is composed of 8 exons and 7 introns within its coding region. These introns are in the same locations as introns in human Gi alpha genes, that encode G proteins closely related to transducin. Primer extension, RNA sequencing, and S1 nuclease protection analyses indicate that the mouse Tr alpha gene transcription start site is 84 bases upstream of the initiation codon. Northern blot analysis shows that the mouse Tr alpha is expressed in the retina, but not in brain, kidney, liver, or heart.

Amino Acid Sequence↗
PubMed1988

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate.

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.

Adenylate Cyclase Toxin↗
PubMed1987

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes.

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.

Adenylyl Cyclases↗
PubMed1987

Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction.

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.

Adenosine Diphosphate Ribose↗
PubMed1985

ADP-ribosylation of transducin by pertussis toxin.

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.

Adenosine Diphosphate Ribose↗
PubMed1983

Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin.

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.

Animals↗