Biomedical subjects
S E Navon
Publications and source records attributed to S E Navon.
Transducin: a signaling switch regulated by guanine nucleotides.
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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.
Characterization of a phosphodiesterase-immunoreactive polypeptide from rod photoreceptors of developing rd mouse retinas.
In the inherited retinal degeneration of rd mice, cyclic GMP accumulates in affected rod photoreceptors prior to their degeneration. A deficiency in the activity of the visual cell phosphodiesterase apparently results in the accumulation of cyclic GMP. The cyclic GMP phosphodiesterase (PDE) of normal mouse photoreceptors is a heteromeric protein complex of about 170 kDa, consisting of the alpha beta catalytic unit and the gamma inhibitory unit. The isolated complex has low enzyme activity but it can be activated by incubation with histone. Affinity-purified polyclonal antibodies against the PDE complex of bovine rod outer segments were prepared and used to identify in retinas of both normal and rd mice PDE-immunoreactive polypeptides which comigrated on SDS-polyacrylamide gels with the large subunits (88 kDa) of the normal PDE complex. During development of normal retinas, the 88 kDa immunoreactive component of the PDE complex were detected by day 7, with immunoreactivity increasing throughout the second postnatal week. In rd retinas, the 88 kDa immunoreactivity increased after 9 postnatal days, decreased during rod photoreceptor degeneration, and was undetectable in mature rd retinas. Under nondenaturing conditions, the PDE-immunoreactive polypeptide of rd retinas sedimented on sucrose gradients with a sedimentation coefficient of 5.6S and an apparent molecular mass of about 105 kDa; no associated histone-activated PDE activity was detected. These findings show that PDE-immunoreactive polypeptides are synthesized in immature rd photoreceptors and that the PDE-immunoreactive polypeptides fail to form a PDE complex which is comparable to that of normal photoreceptors.
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.
Infection of neuroretinal cells in vitro by avian sarcoma viruses UR1 and UR2: transformation, cell growth stimulation, and changes in transducin levels.
Infection in vitro of differentiating chick embryo neuroretinal cells with avian sarcoma viruses UR1 and UR2 results in mitogenic stimulation and morphologic conversion of both support neuronal cells. This was shown by the continuous propagation of transformed cells for over 4 months and growth of reaggregated colonies in liquid medium as well as in soft agar. Production of the transforming proteins p 150 gag-fps and p68 gag-ros of UR1 and UR2, respectively, was similar to that of transformed chick embryo fibroblasts, as judged from in vitro kinase activity assays. The two protein subunits, T beta and T gamma, but not T alpha of the GTP binding protein transducin, found in the retina of many animal species, were present in control neuroretinal cells. Infection with Rous sarcoma virus or UR2 resulted in an inhibition of T gamma synthesis and enhancement of T beta-like protein production.
Immunological determination of transducin content in retinas exhibiting inherited degeneration.
The inherited disorders of rd mice and affected Irish setter dogs are characterized by the accumulation of cyclic GMP (cGMP). Since the cGMP level in normal retinal rods is regulated by a light-activated enzyme cascade involving rhodopsin, transducin, and phosphodiesterase, an abnormality associated with any of these three proteins would cause cGMP accumulation. In order to determine the relationship between different forms of retinal degeneration and the transducin content in the affected retinas, affinity-purified antibodies directed against the individual subunits of bovine transducin were prepared. These antibodies, which recognized transducin in many vertebrate species, were used to compare the retinal content of this protein at various stages of inherited photoreceptor degeneration. In each of the disorders studied (rd and rds mice, RCS rat, and affected Irish setter dog), retinas at early stages of degeneration displayed two characteristics similar to those of normal control retinas. First, all three subunits of transducin were detected and found to have normal electrophoretic mobility, suggesting that these disorders are unlikely to be due to changes in the composition of transducin subunits. Second, the amount of cross-reactive T beta always exceeded those of T alpha and T gamma. This disproportionately higher amount of T beta-like protein became more pronounced as the visual cells degenerated. In retinas which had undergone complete photoreceptor degeneration, cross-reactive T alpha and T gamma were undetectable. In contrast, anti-T beta gamma antibodies detected an amount of T beta-like polypeptide corresponding to 10-25% of the control. Since our anti-T beta gamma antibodies recognize the beta subunit of the GTP-binding N proteins of the adenylate cyclase system, this finding suggests that this residual T beta-like protein, which is not part of transducin, may be associated with other GTP-binding regulatory proteins.
Mapping the binding sites of transducin by sequence homology, chemical modification, and by using monoclonal antibodies.
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Inherited disorders of rd mice and affected Irish setter dogs: evaluation of transducin and cGMP-phosphodiesterase.
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