Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments.

Abstract

Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTPbinding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay ofthe activating capacity ofR*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kuhn, H., Hall, S. W. & Wilden, U. (1984) FEBS Left. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor <5) lightinduced PDEase-activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase.Absorption of light converts rhodopsin into an active form, "photoexcited rhodopsin" (R*), that specifically interacts with three proteins of the rod cell (1). First, a guanine nucleotide-binding protein, transducin,* transiently binds to R* (4). This enables the exchange of GTP for previously bound GDP (3) on the a subunit of transducin (Ta), which then, in its GTP-binding form (Ta-GTP), dissociates from R* (4, 5) and activates a cGMP phosphodiesterase (PDEase) (6). One R* can, during its lifetime, catalyze nucleotide exchange on several hundred transducin molecules (3, 6) and, therefore, lead to the activation of several hundred PDEase molecules (7, 8).Second, a soluble protein kinase, specific for photobleached rhodopsin, binds to R* (9) and phosphorylates it at multiple serine and threonine residues (10, 11). Third, another soluble protein, "48-kDa protein," also binds to bleached disk membranes (9) particularly well if their rhodopsin is phosphorylated (12).Phosphorylation ofrhodopsin has been proposed (13, 14) to function as a "stop" signal, terminating the active state of R* faster than the spontaneous slow decay of the active photoproduct (metarhodopsin II; see refs. 15 and 16) would occur. Speci...

Regulation of α2AR trafficking and signaling by interacting proteins

“…These changes result in activation of the visual G protein, transducin. In addition to activation of molecular pathways that mediate the visual response, signal activation also results in activation of rhodopsin kinase, phosphorylation of rhodopsin, and stabilization of rhodopsin's interaction with arrestin (the "48K antigen") [52][53][54]. Arrestin binding to rhodopsin leads to stabilization of rhodopsin's phosphorylated state and to functional uncoupling from transducin, thus terminating activation.…”

The role of G proteins in transmembrane signalling

“…So far we have been concerned only with G proteins recognizing the cytoplasmic domain of the activated receptor, but those interactions are transient, leaving the domain free to be seen by other intracellular proteins. Two such proteins are rhodopsin kinase (Wilden et al, 1986) and the rather inappropriately named /J-adrenoceptor kinase (Strasser et al, 1986 (Benovic et al, 1989;Lohse et al, 1990). Rodbell (1985) has discussed the considerable potential for modulation of signalling pathways by covalent modification of G proteins, indeed he has coined the term 'programmable messengers' to emphasize their versatile signalling properties.…”

A Segment Corresponding to Amino Acids Val170‐Arg182 of Bovine Arrestin is Capable of Binding to Phosphorylated Rhodopsin

“…Activated transducin in turn stimulates a cyclic GMP phosphodiesterase which decreases the concentration of cyclic GMP in the cytoplasm (Wheeler and Bitensky, 1977;Fung et a]., 1981;Chabre and Deterre, 1989). This causes the closure of cyclic-GMP-gated cation channels in the plasma membrane and results in a hyperpolarization of the rod cell (Cook et al, 1987).The cyclic GMP phosphodiesterase is deactivated when R* becomes phosphorylated by rhodopsin kinase and subsequently binds arrestin (Wilden et al, 1986a;Bennett and Sitaramayya, 1988). Wilden et al (1986a) proposed that arrestin competes with transducin for binding to phosphorylated R* and thereby prevents a further activation of transducin and cyclic GMP phosphodiesterase.…”

“…The cyclic GMP phosphodiesterase is deactivated when R* becomes phosphorylated by rhodopsin kinase and subsequently binds arrestin (Wilden et al, 1986a;Bennett and Sitaramayya, 1988). Wilden et al (1986a) proposed that arrestin competes with transducin for binding to phosphorylated R* and thereby prevents a further activation of transducin and cyclic GMP phosphodiesterase.…”

“…Wilden et al (1986a) proposed that arrestin competes with transducin for binding to phosphorylated R* and thereby prevents a further activation of transducin and cyclic GMP phosphodiesterase. In contrast, Schleicher Abbreviations.…”