Bernard K.-K. Fung

In vivo farnesylation of rat rhodopsin kinase

We have shown by intravitreal injection of [3H]mevalonolactone that a 65 kDa protein in rat photoreceptors is posttranslationally modified by farnesylation. We further identified this 65 kDa prenylated protein as rhodopsin kinase based on its affinity for photolyzed rhodopsin and its ability to autophosphorylate in the presence of [gamma-32P]ATP. The farnesylation of rhodopsin kinase may be important for correctly targeting this enzyme to the photoreceptor outer segments, allowing it to phosphorylate photolyzed rhodopsin efficiently.

The γ subunit of brain G-proteins is methyl esterified at a C-terminal cysteine

The γ polypeptide of brain G‐proteins is carboxyl methylated when the purified βγ subunit complex is reconstituted with S‐adenosyl‐[3H‐methyl]‐L‐methionine and a methyltransferase present in detergent‐stripped brain membranes. By Chromatographic analysis of the 3H‐amino acid generated by exhaustive proteolysis and performic acid oxidation of the 3H‐methylated βγ complex, we show that this modification occurs on the α‐carboxyl group of a C‐terminal cysteine residue. Our result suggests that brain G‐proteins may undergo multiple covalent modification steps, including proteolytic removal of the three terminal amino acids from the predicted common C‐terminal Cys‐Xaa‐Xaa‐Xaa sequence, and the methyl esterification of the resulting terminal cysteine residue. This modification is likely to be associated with lipidation at the sulfhydryl group of the same cysteine, which would explain the tight membrane binding property of the brain βγ complex.

Immological 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.

Analysis of prenylated carboxyl-terminal cysteine methyl esters in proteins

We describe methods that allow for the identification of prenylated and methyl-esterified carboxyl-terminal cysteine residues in proteins radiolabeled in vitro and in vivo . Proteins isotopically labeled in the 15-carbon farnesyl or 20-carbon geranylgeranyl prenyl group, the cysteine residue, or the methyl group are subjected to complete enzymatic hydrolysis under conditions that preserve the methyl ester. The products of this reaction are mixed with synthetic standards of farnesylcysteine, farnesylcysteine methyl ester, geranylgeranylcysteine, and geranylgeranylcysteine methyl ester and are fractionated by reverse-phase HPLC. Each of these compounds is readily resolved, and the product identification can be confirmed using normal-phase thin-layer chromatography. Further confirmation of the linkage of the methyl group to the α -carboxyl group of a carboxyl-terminal cysteine residue can be obtained by identifying radiolabeled cysteic acid methyl ester derived from performic acid-oxidized samples of digests of cysteine or methyl ester labeled material. Cysteic acid methyl ester can be identified by amino acid analysis on cation-exchange resin as well as by thin-layer chromatography. We describe synthetic methods for preparing the farnesyl- and geranylgeranylcysteine derivatives as standards, as well as general methods for detecting radiolabeled methyl esters.

Transducin: a signaling switch regulated by guanine nucleotides.

Publisher Summary This chapter discusses the information obtained from the biochemical studies of transducin and attempts to integrate this information with the proposed structure of the GTP binding α subunit of transducin (T α ). One of the best-studied biological processes regulated by G proteins is the visual excitation of vertebrate photoreceptors, in which the direct involvement of transducin in coupling photolyzed rhodopsin to cGMP phosphodiesterase (PDE) has been conclusively demonstrated. As all the members of the G protein family share a common structural motif and appear to exhibit a similar mode of action, it is generally believed that the biochemical pathway elucidated in the visual system will help to provide a better understanding of the role of G proteins in other less well-defined signal transduction processes. Subsequently, important progress has been made in recent years, especially in the characterization of the GTP binding regulatory proteins. Thereafter, the involvement of nucleotide-binding protein, the existence of tightly bound nucleotides, and their exchange-hydrolysis cycles may represent a common motif in controlling multisubunit enzymes in cellular regulation.