The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.

Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyllmethionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the a subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory y subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the a cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the a-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.Visual excitation in vertebrate retinal rods is mediated by a cGMP cascade that converts light into cellular responses (reviewed in ref. 1). An amplified signal is generated when photolyzed rhodopsin activates a retinal G protein (transducin), which, in turn, stimulates the activity of a cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE). PDE, the key regulatory enzyme involved in this process, is composed of a (Mr = 90,000), 13 (Mr = 88,000), and y (Mr = 10,000) polypeptides. In this multimeric form, the hydrolytic activity associated with a and , is inhibited by the y subunit. PDE is activated when 'y is removed by interaction with the GTP-bound form of transducin. The transient decrease in intracellular concentration of cGMP then leads to the closure of many cGMP-regulated cation channels in the plasma membrane, resulting in a decrease in Na+ influx and hyperpolarization of the rod.PDE is a peripheral membrane protein that can be eluted from disk membranes of the outer segments by extraction with low ionic strength buffers (2, 3) or by brief proteolysis with trypsin (4). However, unlike transducin, which is known to bind tightly to rhodopsin in a light-dependent manner, PDE appears to be associated with the lipid matrix (5). The mechanism by which PDE attaches to the surface of the disk membrane, however, remains to be elucidated.Recently, we proposed that a class of membrane-associated proteins is modified by methyl esterification at the a-carboxy...

No abstract

No abstract