cDNA cloning and characterization of a novel squid rhodopsin kinase encoding multiple modular domains

Abstract

Rhodopsin phosphorylation is one of the key mechanisms of inactivation in vertebrate and invertebrate visual signal transduction. Here we report the cDNA cloning and protein characterization of a 70-kDa squid rhodopsin kinase, SQRK. The cDNA encoding the 70-kDa protein demonstrates high sequence identity with octopus rhodopsin kinase (92%) and mammalian β-adrenergic receptor kinases (63–65%), but only 33% similarity with bovine rhodopsin kinase, suggesting that invertebrate rhodopsin kinases may be structurally similar to β-adrenergic receptor kinases. This cDNA encodes three distinct modular domains: RGS, S/TKc, and PH domains. The native SQRK is an eye-specific protein that is only expressed in photoreceptor cells and the optic ganglion as determined by immunoblotting. Purified SQRK is able to phosphorylate both squid and bovine rhodopsin. Squid rhodopsin phosphorylation by purified SQRK was sensitive to both Mg2+ and GTPγS but was insensitive to Ca2+/CaM regulation. The ability of SQRK to phosphorylate rhodopsin was totally lost in the presence of SQRK-specific antibodies. Our results suggest that SQRK plays an important role in squid visual signal termination.

Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase

“…1996; Kahn and Matsumoto 1997). In previous reconstitution studies, we observed no effects of calmodulin, or inhibitors of Ca 2+ /calmodulin‐dependent protein kinase II or protein kinase C on light‐dependent phosphorylation of Loligo arrestin (Mayeenuddin and Mitchell 2001). Instead, antibodies raised against recombinant squid rhodopsin kinase (SQRK) were able to abrogate arrestin phosphorylation, suggesting that squid arrestin and rhodopsin are both SQRK substrates.…”

“…1997). It is possible that a greater inhibitory effect would have been observed in squid rhabdomeric membranes if rhodopsin phosphorylation had been increased; however, as SQRK contains an RGS domain and itself has some GTPase enhancing activity (Mayeenuddin and Mitchell 2001), this could not be demonstrated within the limitations of these assays. In fact, a functional role for rhodopsin phosphorylation in termination of visual signaling has yet to be demonstrated in invertebrates.…”

“…Kinase assays were carried out as originally described (Vandenberg and Montal 1984), with modifications as previously described (Mayeenuddin and Mitchell 2001). Briefly, rhabdomeric membranes (10 μg) were incubated with SQRK (1 μg) and purified arrestin (1 μg) for 20 min at 26°C in kinase assay buffer (50 mmol/L Tris, pH 8.0, 15 mmol/L MgSO 4 , 1 mmol/L EGTA, 1 mmol/L ATP, 2 mmol/L DTT, 100 mmol/L ouabain, and 1 μmol/L GTPγS) with [γ 32 P]ATP (400 000 cpm/μL).…”

Degradation of the non-palmitoylated invertebrate visual guanine-nucleotide binding protein, iGqα(C3,4A), by the ubiquitin-proteasomal pathway is regulated by its activation and translocation to the cytoplasm

“…Squid eyes have been used by our laboratory and others as a rich source of visual signaling proteins to isolate and characterize many components of the cephalopod visual system~Ryba et Mitchell et al, 1995;Suzuki et al, 1995;Narita et al, 1999;Mayeenuddin & Mitchell, 2001, 2003!. Invertebrate iG q a subunits are very similar to mammalian G q a~Ryba et al, 1993; Go & Mitchell, 2003!…”

The effect of phosphorylation on arrestin–rhodopsin interaction in the squid visual system

“…) and Loligo pealei (Mayeenuddin and Mitchell ). We have previously determined that squid ( Loligo pealei ) visual arrestin binds metarhodopsin in a light‐dependent manner and that both metarhodopsin and arrestin are phosphorylated by the squid rhodopsin kinase (SQRK) (Mayeenuddin and Mitchell , ; Swardfager and Mitchell ). The sites of squid arrestin phosphorylation have yet to be determined; however, other invertebrate arrestins and the non‐visual mammalian arrestins (β‐arrestins) are also phosphorylated by kinases at one or more serine and/or threonine residues near the C‐terminus (Lin et al .…”