Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence.

Abstract

Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes G, and G1, the regulatory proteins of adenylate cyclase. Here we report the cDNA sequence and deduced amino acid sequence of transducin's a subunit (Ta). The cDNA was isolated, by screening with an antibody probe, from a bovine retinal cDNA library in the expression vector Xgtll. The 2.2-kilobase cDNA insert hybridized to a single 2.6-kilobase poly(A)+ RNA species present in extracts of bovine retina but not of bovine heart, liver, or brain. The nucleotide sequence of the cDNA revealed an open reading frame long enough to encode the entire 39-kDa T. polypeptide. The polypeptide sequence deduced from the cDNA would be composed of 350 amino acids and have a molecular weight of 39,971. Portions of the sequence matched reported amino acid sequences of Ta tryptic fragments, including sites specificlly ADP-ribosylated by cholera and pertussis toxins. The predicted sequence also includes four segments, ranging from 11 to 19 residues in length, that exhibit significant homology to sequences of GTP-binding proteins, including the ras proteins of man and yeast and the elongation factors of ribosomal protein synthesis in bacteria, EF-G and EF-Tu. In combination with previous functional studies of tryptic fragments of Ta, the deduced amino acid sequence makes it possible to predict which portions of the polypeptide interact with other molecules involved in retinal phototransduction.Transducin, a guanine nucleotide binding protein of retinal rod cells, mediates the activation of a cyclic GMP phosphodiesterase (PDEase) in response to photoexcitation of rhodopsin (1). Each photolyzed rhodopsin (R*) molecule activates hundreds of transducin molecules by catalyzing the exchange of GTP for GDP at transducin's guanine nucleotide binding site. The activated transducin-GTP complex in turn stimulates the hydrolysis of many cGMP molecules by PDEase. Hydrolysis of bound GTP returns transducin to its inactive state.Transducin contains three subunits, a, ,B, and y, whose sizes are 39, 35, and =8 kDa, respectively. Transducin exhibits striking structural and functional homologies to the stimulatory (Gs) and inhibitory (Gi) coupling proteins of hormone-sensitive adenylate cyclase. Each protein is a heterotrimer in which the respective subunits exhibit similar amino acid compositions and proteolytic peptide maps (2). In each case, the protein's interaction with an excited signal detector (rhodopsin or hormone receptor) triggers dissociation of the /3y complex from the a subunit, which then alters activity of the effector molecule (PDEase or adenylate cyclase) (1, 3).After hydrolysis of GTP bound to the a subunit, the P-y

Why photoreceptors die (and why they don't)

“…(18)(19)(20) As in other such cascades, the binding of the G protein to the receptor causes the exchange of GTP for GDP on the guanylyl nucleotide binding site of the transducin a subunit (T a ). The T a -GTP then separates from the T b and T g subunits of the transducin and binds to the effector enzyme of the cascade, a photoreceptor-specific cyclic nucleotide phosphodiesterase (PDE).…”

rasH mutants deficient in GTP binding.

“…The results presented here are consistent with these predictions and additionally suggest that Lys-117 is important for guanine nucleotide binding. In addition to the rasH amino acid sequence 110 through 120, sequence alignment comparisons identified three other principal regions of significant homology between ras and other GTP-binding proteins, including transducin and the bacterial elongation (EF-Tu and EF-G) and initiation (IF2) factors (15,23,36). These three regions of homology are represented by the cellular ras amino acid residues (i) 5 to 15, (ii) 55 to 65, and (iii) 72 to 85, and they appear in the same order in a-transducin and the bacterial elongation factors EF-Tu and EF-G.…”

Primary structure of bovine cerebellum GTP‐binding protein G39 and its effect on the adenylate cyclase system

“…CAMP level stabilization in neurons in cases of sharp increase of blood catecholamine concentration, for example, under stress conditions. A number of authors hold an opinion based on indirect indications that the G-protein &-subunit interacts with the N-terminal region of a-subunits [22,23]. However, when comparing G39 structure with structures of G,, Gi, T,i and Trr2, we noted a comparatively high variability in the N-terminal fragments of these proteins.…”