A novel complex from bovine visual cells of a 33,000-dalton phosphoprotein with .beta.- and .gamma.-transducin: purification and subunit structure

Abstract

Photoreceptors of mammalian retinas contain a 33-kDa (33K) protein that is phosphorylated, in vitro, by cyclic nucleotide dependent protein kinases. The 33K protein is phosphorylated in the dark, in situ, and dephosphorylated upon illumination. The soluble 33K protein from bovine retinas has been purified to near homogeneity by extraction at pH 5.7 and chromatography on ion-exchange, gel filtration, and hydroxylapatite columns. In the native conformation, the 33K protein is associated with a 37-kDa (37K) and a 10-kDa (10K) protein, forming a trimeric complex with a sedimentation coefficient of 4.9 S and an apparent molecular mass of 77 kDa. The 33K protein can be dissociated from the 37K/10K complex by centrifugation in the presence of high pH and high salt; the subunits reassociate to form the trimeric complex upon recentrifugation in an isotonic buffer with neutral pH. The 33K protein is phosphorylated rapidly by exogenous kinase, in vitro, whereas the 37K and 10K subunits remain unphosphorylated. The 37K and 10K subunits cross-react with antibodies prepared against the beta- and gamma-subunits, respectively, of bovine transducin, indicating that the 37K and 10K subunits are immunologically identical with beta- and gamma-transducin, respectively. No immuno-cross-reactivity was observed between the 33K protein and an antibody against the alpha-subunit of bovine transducin. The 33K-beta-/gamma-transducin complex exhibits striking similarity to transducin in its subunit structure and mode of subunit interaction, suggesting it may play an important role in the metabolism and function of rod photoreceptor cells.

Site-specific Phosphorylation of Phosducin in Intact Retina

“…Physiological Implications-The results presented here give further insight into the possible physiological importance of the Pdc/G t ␤␥ interaction, which has remained elusive since its initial discovery nearly two decades ago (10). The data show that the binding of Pdc to G t ␤␥ is elegantly linked to the light adaptation state of the photoreceptor cell through phosphorylation and dephosphorylation events that are tied to Ca 2ϩ and cAMP concentrations that are in turn controlled by light (31)(32)(33).…”

“…In contrast, the only known function of the G t ␤␥ subunit complex is to mediate the interaction of G t ␣ with rhodopsin, yet in other G protein signaling systems G␤␥ plays significant roles in downstream effector activation (9). Besides G t ␣, the only other reported binding partner for G t ␤␥ in photoreceptor cells is phosducin (Pdc), a 28-kDa phosphoprotein expressed at high levels in both photoreceptor cells and pinealocytes (10,11). Pdc is phosphorylated in dark-adapted photoreceptors and dephosphorylated in response to light (12).…”

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…Interest in Pdc increased significantly when it was shown to co-purify from retinal extracts with the βγ subunit complex of the retinal G protein, transducin (G t βγ) [11]. These observations resulted in the protein being given the name phosducin.…”

“…These GTPase accelerating proteins (GAPs) play a vital role in determining the lifetime of the G protein signal [10]. At the level of Gβγ, the binding of Pdc [11][12][13][14] and PhLP1 [15,16] to Gβγ has been proposed to control the amount of Gβγ available for interaction with effectors or with Gα-GDP. However, this Gβγ sequestration model for Pdc and PhLP1 function has been brought into serious question by recent findings [17][18][19].…”

“…The family can be divided into three subgroups [20]. Subgroup I includes the initial members of the family, Pdc and PhLP1, which have been shown to bind Gβγ subunits with high affinity [11,21,22]. Pdc expression is very restricted, being found at significant levels in only the photoreceptor cells of the retina and in the pineal gland [11,23].…”