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PubMed1998

Functional heterogeneity of transducin alpha subunits.

The N-terminal glycine of transducin alpha subunits is acylated by lauroyl (C12:0), myristoyl (C14:0), (cis-delta5)-tetradecaenoyl (C14:1) or (cis,cis-delta5,delta8)-tetradecadienoyl (C14:2) fatty acyl groups. We examined functional heterogeneity of transducin by sequentially eluting it from bleached outer segments using increasing concentrations of GTP then identifying the N-terminal acyl groups on the eluted alpha subunits. C14:2 acylated transducin eluted at low GTP concentrations followed by C12:0, C14:1 and C14:0 transducin at higher GTP concentrations. This suggests functional heterogeneity in the different forms of transducin alpha subunits.

Acetylation↗
PubMed1994

Circular dichroism of metaiodopsin II and its binding to transducin: a comparative study between meta II intermediates of iodopsin and rhodopsin.

Through low-temperature absorption and circular dichroism (CD) spectroscopies, and G-protein (transducin) binding experiments, we have investigated molecular properties of the meta II intermediate of iodopsin, a cone visual pigment present in chicken red-sensitive cones. The meta II intermediate of iodopsin (metaiodopsin II, lambda max = 390 nm) displayed a positive CD band at about 390 nm and a large negative CD band below 300 nm. It dissociated into all-trans-retinal and the protein moiety. A long-lived intermediate corresponding to the meta III intermediate of rhodopsin was not observed in iodopsin, under our experimental conditions. Decay of metaiodopsin II was significantly suppressed in the presence of transducin, but not in the presence of both transducin and GTP, indicating that metaiodopsin II can interact with transducin and activate it. Both metaiodopsin II and metarhodopsin II displayed a large negative CD band below 300 nm. This fact suggested that during the formation of both meta II intermediates, some aromatic amino acid residues and/or a disulfide bond are rearranged, which may be important for expression of catalytic activity for exchange of GDP to GTP on transducin. On the other hand, metaiodopsin II decayed more than 10 times faster than metarhodopsin II. This fact may be one of the reasons why cones are less photosensitive than rods.

Animals↗
PubMed1998

Incorporation of rhodopsin in laterally structured supported membranes: observation of transducin activation with spatially and time-resolved surface plasmon resonance.

Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.

Biosensing Techniques↗
PubMed1998

Interaction between photoactivated rhodopsin and the C-terminal peptide of transducin alpha-subunit studied by FTIR spectroscopy.

Structural changes in the complex formed between photolyzed bovine rhodopsin and a synthetic 11-mer peptide corresponding to the C-terminal region of the transducin alpha-subunit (Gtalpha) were analyzed by means of Fourier transform infrared spectroscopy. A complex with a protonated Schiff base appears at the beginning, accompanying the formation of an alpha-helix. This complex evolves into another which abolishes the original structure but retains the protonated Schiff base. This complex exhibits the same spectral shape as that of the final stable complex with an unprotonated Schiff base. The Fourier transform infrared spectrum for the formation of this final complex was compared to that with transducin [Nishimura, S., Sasaki, J., Kandori, H., Matsuda, T., Fukada, Y., and Maeda, A. (1996) Biochemistry 35, 13267-13271]. A large part of the frequency shifts of the peptide carbonyl vibrations which form upon complex formation with transducin but are absent with the synthetic 11-mer peptide must be structural changes in other sites, such as the nucleotide binding site in Gtalpha. The peptide, like transducin, shows the perturbation of a carboxylic acid in an extremely apolar environment. Some of the changes in the peptide backbone remain in the complex formed with the peptide. These are due to sites where rhodopsin interacts with the C-terminal region of Gtalpha. Specifically, the labeling of the peptide amide corresponding to Leu349 of transducin by 15N reveals weakening of the hydrogen bond of the peptide N-H of Leu349 and/or distortion of a peptide bond between Gly348 and Leu349 upon complex formation.

Amides↗
PubMed1990

Subsecond deactivation of transducin by endogenous GTP hydrolysis.

The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.

Animals↗
PubMed1986

C-terminal peptides of rhodopsin. Determination of the optimum sequence for recognition of retinal transducin.

In vertebrate retinal rod outer segments, transducin, a guanine-nucleotide-binding protein, mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase. Whereas the T alpha subunit (39 kDa) of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunits (35 and 10 kDa) may function to physically link T alpha with photolysed rhodopsin. We have previously reported that a site of binding of transducin is on the C-terminus of bovine rhodopsin. By using competition with synthetic peptides, the recognition region was localized to bovine opsin amino acid residues 317-339. Further studies are detailed which determine the boundaries of this binding site on rhodopsin, as well as some of the critical amino acids needed for transducin binding. These results suggest that the serine and threonine residues in the rhodopsin C-terminal peptides Rhod-1 and Rhod-3 are critical for reconstitution of transducin GTPase activity.

Amino Acid Sequence↗
PubMed1984

Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin.

We have isolated and characterized a cDNA clone that encodes the gamma subunit of transducin, the guanine nucleotide binding regulatory protein found in vertebrate photoreceptors. The gamma subunit was separated from the alpha and beta subunits of transducin and purified to homogeneity by reversed-phase high performance liquid chromatography. The sequence of the first 45 amino acids at the amino terminus of this polypeptide was then determined by automated Edman degradation. Oligodeoxynucleotide probes corresponding to two nonoverlapping regions of this sequence were synthesized and then used to screen a bovine retinal cDNA library. One probe, T gamma 1, was a mixture of 32 different heptadecamers complementary to all possible mRNA sequences that could encode a portion of the T gamma sequence; the other probe, T gamma 2, was a mixture of 128 different heptadecamers. Thirteen clones that hybridized with T gamma 1 were selected. Only one of these had an insert that also hybridized with T gamma 2. The DNA sequence of this insert encodes a 73-amino acid polypeptide that corresponds to the transducin gamma subunit on the basis of amino-terminal sequence, amino acid composition, and carboxyl-terminal sequence. The molecular weight of the mature gamma subunit is 8400. It appears to be synthesized as a discrete polypeptide and not as a domain of a larger precursor polyprotein. The transducin gamma subunit is very hydrophilic and acidic; it has 19 acidic and 11 basic amino acids as well as three cysteine residues. Furthermore, significant homology was found in comparisons of the nucleic acid sequence corresponding to the carboxyl terminus of the gamma transducin transcript with the sequences corresponding to the carboxyl terminus of ras oncogene products, suggesting a possible ancestral relationship between these genes.

Amino Acid Sequence↗
PubMed1995

Transducin activation by the bovine opsin apoprotein.

The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.

Animals↗
PubMed2000

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites.

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.

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

Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells.

Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity binding to Gtbetagamma and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gbetagamma of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.

Animals↗
PubMed2005

Transducin activation state controls its light-dependent translocation in rod photoreceptors.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.

Adaptation, Ocular↗
PubMed1992

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors.

Transducin is a guanyl nucleotide-binding protein that couples rhodopsin photolysis to hydrolysis of guanosine 3',5'-monophosphate in rod photoreceptor cells of vertebrate retinas. Several complementary DNA clones encoding transducin subunits have recently been characterized. One clone, isolated from a bovine retina complementary DNA library, encodes a previously unidentified polypeptide with an amino acid sequence 78% identical to the sequence of the alpha subunit of bovine rod outer segment transducin. Antibodies to a synthetic peptide with amino acid sequence derived specifically from this novel polypeptide recognize a 41-kilodalton polypeptide in homogenates of bovine retina. Localization of this polypeptide in bovine retina by indirect immunofluorescence demonstrates that it is expressed only in cone outer segments. Antibodies to specific sequences found only in the rod transducin alpha subunit recognize a polypeptide localized only in the rod outer segment. Therefore, bovine rod and cone cells each express structurally related yet significantly different forms of transducin.

Animals↗
PubMed2005

The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors.

PURPOSE: Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS: Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS: LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS: Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.

Animals↗
PubMed2003

Light stimulates a transducin-independent increase of cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mutant nof.

Transducins couple visual pigments to cGMP hydrolysis, the only recognized phototransduction pathway in vertebrate photoreceptors. Here we describe a zebrafish mutant, no optokinetic response f(w21) (nof), with a nonsense mutation in the gene encoding the alpha subunit of cone transducin. Retinal morphology and levels of phototransduction enzymes are normal in nof retinas, but cone transducin is undetectable. Dark current in nof cones is also normal, but it is insensitive to moderate intensity light. The nof cones do respond, however, to bright light. These responses are produced by a light-stimulated, but transducin-independent, release of Ca2+ into the cone cytoplasm. Thus, in addition to stimulating transducin, light also independently induces release of Ca2+ into the photoreceptor cytoplasm.

Adaptation, Ocular↗
PubMed2003

Light-dependent redistribution of visual arrestins and transducin subunits in mice with defective phototransduction.

PURPOSE: The light-dependent redistribution of phototransduction components in photoreceptor cells plays a role in light adaptation. Upon illumination, rod and cone arrestins (Arr and cArr) translocate from the inner to the outer segments while transducin subunits (Talpha, Tbetagamma) translocate in the opposite direction. The underlying translocation mechanisms are unclear. This study examines these previously demonstrated translocation in mice with defective phototransduction. METHODS: The distribution of Arr, cArr, Talpha, and Tbetagamma was examined using immunoblotting and immunocytochemistry in dark- and light-adapted single knockout mice lacking G-protein coupled receptor kinase 1 (Grk1-/-) and double knockout mice lacking GRK1 and transducin alpha subunit (Grk1-/-/Gnat1-/-), or lacking GRK1 and arrestin (Grk1-/-/Arr-/-). RESULTS: Arr redistributed in the light to the outer segments in Grk1-/- mice as well as in Grk1-/-/Gnat1-/- double knockout retinas. Immunoblotting revealed that approximately 25-50% of Arr associated with the membrane in light-adapted wild-type, Grk1-/- and Gnat1-/-/Grk1-/- mouse retinas. In contrast, cArr did not stably associate with light-adapted membranes in either wild-type or Grk1-/- retinas under our experimental conditions, but redistributed to the cone outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurred in both wild-type and Grk1-/-/Arr-/- double knockout photoreceptors. However, Tbetagamma subunits did not redistribute in the absence of Talpha, suggesting that transducin only translocates as an intact heterotrimer. CONCLUSIONS: We conclude that in rods, Arr redistribution requires neither rhodopsin phosphorylation nor phototransduction, suggesting the presence of another light-dependent pathway to trigger translocation. In cones, the light-dependent movement of cArr appears to be independent of stable association with the cone pigments. The light-dependent translocations of Arr and transducin subunits in opposite directions appear to be based on independent mechanisms.

Animals↗
PubMed1990

Sites of interaction in the complex between beta- and gamma-subunits of transducin.

Transducin, the guanine nucleotide-binding regulatory protein in rod outer segments, is a heterotrimer consisting of alpha-, beta-, and gamma-subunits. Activation of the photoreceptor, rhodopsin, by light, results in activation of transducin which cleaves to form transducin alpha. GTP and a complex of beta gamma-subunits. We have investigated the point(s) of contact between the subunits of transducin by analyzing for the formation of intersubunit disulfide bond(s) in the presence of copper phenanthroline. The formation of a new species with an apparent molecular mass of 43 kDa was observed which had resulted from the formation of a disulfide bond between the beta- and gamma-subunits. The amino acid residues participating in the disulfide bond were identified as Cys-25 in the beta-subunit and Cys-36 and/or Cys-37 in the gamma-subunit. Thus, these cysteine residues and, probably, some of the adjacent amino acid residues form a point of contact between the beta- and gamma-subunits of transducin in the stable complex.

Amino Acid Sequence↗
PubMed1988

Characterization of transducin from bovine retinal rod outer segments. Use of monoclonal antibodies to probe the structure and function of the subunit.

A panel of monoclonal antibodies has been developed against the T alpha, T beta and T gamma subunits of bovine transducin. Two anti-T alpha antibodies from this panel (TF15 and TF16) and a third one (4A) against frog T alpha (Witt, P. L., Hamm, H. E., and Bownds, M. D. (1984) J. Gen. Physiol. 84, 251-263) were characterized. Each of these monoclonal antibodies recognizes a different region of T alpha and has a specific effect on the function of transducin. The binding of TF15 is reversibly enhanced by treating T alpha with either 1 M guanidinium chloride or, to a smaller extent, by the removal of bound guanine nucleotide. Its epitope is located in a 12-kDa tryptic fragment containing the binding site for the guanine moiety of GTP. Taken together, these results support previous observations that the conformation of T alpha is modulated by the occupancy of the guanine nucleotide binding site. In contrast to TF15, TF16 recognizes only the native form of T alpha. Its epitope resides within the central portion of the T alpha molecule. While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin. These effects are unlikely to be caused by the occupation of the guanine nucleotide binding site by TF16 because this antibody quantitatively precipitates T alpha-GTP gamma S. We propose that bound TF16 locks T alpha in a conformation that prevents the entrance of guanine nucleotide and favors T beta gamma association. In contrast to TF16, the epitope of 4A was mapped to the amino-terminal region of T alpha. This monoclonal antibody blocks pertussis toxin-catalyzed ADP-ribosylation, GTP gamma S uptake, and T alpha-T beta gamma association. Moreover, the binding site for 4A becomes inaccessible when transducin binds to photolyzed rhodopsin. These results suggest that the inhibitory effects of 4A are due to a simultaneous steric blockage of both the interaction of T alpha with T beta gamma and their binding to photolyzed rhodopsin. The results obtained from these studies are correlated with the structure and function of T alpha.

Animals↗