Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors: Biochemical identification and characterization of new subunits

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of α (39,000), β (36,000), and γ (∼7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct α and two distinct βγ activities in frog (Rana catesbiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these α fractions was proportional to the amount of the M_r. 39,000 protein. The first α was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but α subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second αs were the same protein; however, the third α showed different characters as follows. We designated α in the first two fractions as α1, and α in the third fraction as α2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both α subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/ mol of α. Compared with α1, α2 required larger amounts of rhodopsin and βγ for the binding of a hydrolysis-resistant GTP analog. α2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both α1 and α2 complexed with βγ or βδ (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two βγ fractions revealed that the main fraction was composed of a βγ complex; however, the second active fraction was composed of β complexed with δ (M_r 12,000). Compared with βγ, βδ stimulated GTP binding to α1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five β and two γ isoforms in βγ. Only one β isoform was present in βδ. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.