Rhodopsin kinase activity modulates the amplitude of the visual response in Drosophila Seung-Jae Lee*, Hong Xu, and Craig Montell† Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved June 30, 2004 (received for review March 29, 2004) A feature shared between Drosophila rhodopsin and nearly all tractability of fly genetics (14). As in mammals, light-activated other G protein-coupled receptors is agonist-dependent protein rhodopsin is phosphorylated and interacts with a protein, arres- phosphorylation. Despite extensive analyses of Drosophila photo- tin, which facilitates deactivation of the receptor. However, transduction, the identity and function of the rhodopsin kinase unlike mammalian phototransduction, light activation in Dro- (RK) have been elusive. Here, we provide evidence that G protein- sophila is coupled to stimulation of phospholipase C rather than coupled receptor kinase 1 (GPRK1), which is most similar to the a cGMP-phosphodiesterase. ␤-adrenergic receptor kinases, G protein-coupled receptor kinase 2 Two Drosophila genes encoding putative GRKs (GPRK1 and (GRK2) and GRK3, is the fly RK. We show that GPRK1 is enriched in GPRK2) were isolated more than a decade ago (15), but were photoreceptor cells, associates with the major Drosophila rhodop- dismissed as candidate RKs due in part to their much greater sin, Rh1, and phosphorylates the receptor. As is the case with similarity to mammalian nonvisual GRKs than to mammalian mammalian GRK2 and GRK3, Drosophila GPRK1 includes a C- RKs. Subsequent analysis of GPRK2 demonstrated that it is terminal pleckstrin homology domain, which binds to phosphoi- expressed in the ovaries and required for egg morphogenesis nositides and the G␤␥ subunit. To address the role of GPRK1, we (16). No additional GRK-related genes appear to be encoded in generated transgenic flies that expressed higher and lower levels flies. Although the Drosophila RK gene has not been identified, of RK activity. Those flies with depressed levels of RK activity RK activities has been characterized biochemically in larger fly displayed a light response with a much larger amplitude than WT. species (17, 18). RKs have been cloned from octopus and squid Conversely, the amplitude of the light response was greatly sup- and shown to bear much greater similarity to the ␤-adrenergic pressed in transgenic flies expressing abnormally high levels of RK receptor kinases, GRK2͞3 and GPRK1, than to mammalian RKs activity. These data point to an evolutionarily conserved role for (19, 20). However, the physiological roles of rhodopsin phos- GPRK1 in modulating the amplitude of the visual response. phorylation by these or any other invertebrate RK are not known. protein-coupled receptors (GPCRs) are critical for a In the current article, we provide evidence that GPRK1 is the Gdiversity of processes, which in excitable cells range from Drosophila RK. Consistent with this conclusion, we found that vision to analgesia, neuronal differentiation, cardiac function, GPRK1 was enriched in photoreceptor cells, interacted with rhodopsin, and phosphorylated the major rhodopsin. In addi- and synaptic transmission. A key aspect of GPCR signaling is the ␤␥ ability to rapidly adjust receptor activity in response to agonist tion, GPRK1 bound to PIs as well as to the G subunit. Of stimulation. Such agonist-dependent modulation appears to be primary importance here, we found that transgenic flies that accomplished in part through phosphorylation of the receptor displayed a decrease in RK activity in vivo exhibited a much (1–4). Mammals express Ͼ1,000 GPCRs; yet, the human and larger photoresponse than WT. Conversely, the amplitude of the rodent genomes appear to encode only seven G protein-coupled photoresponse was diminished in transgenic flies displaying receptor kinases (GRKs or GPRKs) (3). higher levels of RK activity. We conclude that the physiological GRKs fall into three subgroups, one of which includes the two function of the Drosophila RK is to modulate the amplitude of visual GRKs, GRK1 and GRK7 (1–3). The two nonvisual GRK the light response. Given the known roles, biochemical charac- subgroups consist of GRK2͞GRK3, which phosphorylate the teristics, and domain organizations of mammalian GRKs, our ␤ ͞ ͞ results point to striking similarities between GPRK1 and mam- -adrenergic receptor, and GRK4 GRK5 GRK6. GRKs in- ␤ clude a central catalytic region and an N-terminal RGS-like malian -adrenergic receptor kinases. domain, but differ primarily in the C-terminal region. A distin- Materials and Methods guishing feature of the GRK2͞GRK3 is a C-terminal pleckstrin Fly Stocks and Germ-Line Transformation. The following fly strains homology (PH) domain, which binds both phosphoinositides were reared at 25°C under a 12-h light͞12-h dark cycle without (PIs) and the G␤␥ subunit. In addition, GRK2͞GRK3 bind the or with heat shock (30-min heat shocks at 37°C twice per day for G␣ subunit through the N-terminal RGS-like domain, which ϩ q 9 days) as indicated: cn bw (wt), cn bw P[w , hs-gprk1](ogprk1), inhibits the interaction between G␣ and phospholipase C␤ (5). ϩ q cn bw;P[w , hs-gprk1K220R](ogprk1K220R), sine oculis (so), rdgAP38 Interestingly, some functions of GRKs appear to be independent (rdgA), w;;ninaEP332 (ninaE), yw;;rdgC306 (rdgC), and ry506 of the catalytic activity (6), which could potentially be mediated ϩ P[ry ,Gprk26936](gprk2). The DNA constructs used for the through the N- or C-terminal domains. germ-line transformation and other assays in this study are The biological roles of GRKs have been characterized in a described in Supporting Materials and Methods, which is pub- variety of systems. The first GRK to be identified is rhodopsin lished as supporting information on the PNAS web site. kinase (RK; also GRK1) (7), and mutations in this enzyme cause defects in the kinetics of deactivation and an increase in the amplitude of the light response (8–10). Defects in the activities This paper was submitted directly (Track II) to the PNAS office. of nonvisual GRKs, typically result in reductions in desensitiza- Abbreviations: PH, pleckstrin homology; GRK (or GPRK), G protein-coupled receptor kinase; tion (2), the phenomenon by which signaling is diminished upon PI, phosphoinositide; RK, rhodopsin kinase; IP, immunoprecipitation; ERG, electroretino- prolonged or repeated exposure to an agonist. Mutations in both gram; TRP, transient receptor potential. visual and nonvisual GRKs can also result in supersensitivity to *Present address: Department of Biochemistry and Biophysics, Mission Bay Genetech Hall, agonist stimulation (10–13). University of California, San Francisco, CA 94143. Drosophila visual transduction has served as a paradigm to †To whom correspondence should be addressed. E-mail: [email protected]. characterize G protein-coupled neuronal signaling, owing to the © 2004 by The National Academy of Sciences of the USA 11874–11879 ͉ PNAS ͉ August 10, 2004 ͉ vol. 101 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402205101 Downloaded by guest on October 2, 2021 Northern Blots, Western Blots, and Coimmunoprecipitation (Co-IP). gprk1 and rp49 DNAs were labeled with [␣-32P]dATP and used to probe Northern blots containing 5 ␮g poly(A)ϩ mRNAs. For Western blots, 20 heads or three bodies (per 100 ␮l) were homogenized in TBS [20 mM Tris͞150 mM NaCl͞1ϫ protease inhibitor mixture (Sigma), pH 7.5] containing 1% Triton X-100 (TBST) and centrifuged for 5 min at 18,000 ϫ g. The superna- tants were fractionated by SDS͞PAGE, transferred to a poly- (vinylidene difluoride) membrane, and probed with ␣-GRK2 antibodies. A description of the antibodies is included in Sup- porting Materials and Methods. Signals were detected by using an ECL kit (PerkinElmer) or 125I-labeled protein A (NEN). Sep- arate blots containing the same extracts were probed with the ␣-transient receptor potential (TRP) or ␣-myc antibodies. For quantification (see Fig. 2), the membranes were exposed to a BAS-III imaging plate with a PhosphorImager (BAS-1500, Fuji Film). Co-IPs were performed as described (21) with modifica- tions (see Supporting Materials and Methods). In Situ Hybridizations. In situ hybridizations on sections of adult fly heads were performed as described (21). Phosphorylation of Rh1. Five fly heads added to 100 ␮l of buffer ͞ ͞ A (TBS plus 1 mM DTT 3 mM MgCl2 1 mM EGTA) were illuminated for 20 sec with blue light and homogenized under a red photographic safety light, and an additional 100 ␮l of buffer A containing 0.1 mM [␥-32P]ATP (ICN) was added. The ho- mogenates were incubated for 10 min at 25°C in the dark and NEUROSCIENCE Fig. 1. Expression pattern of gprk1.(A) Northern blot analysis. mRNAs from centrifuged at 20,000 ϫ g for 10 min. The pellets were washed ␮ ␮ WT heads (WH), sine oculis heads (SH), and WT bodies (WB) were probed with once with 500 l of buffer A, dissolved in 30 l of SDS sample 32 ͞ P-labeled gprk1 and rp49 DNAs. RNA markers (kb) are indicated. (B) Western buffer, and fractionated by SDS PAGE. The dried gels were blot analysis. ␣-GRK2 antibodies were used to probe a blot containing extracts analyzed by autoradiography or with a PhosphorImager. West- from WT bodies (WB), WT heads (WH), sine oculis heads (SH), and heads from ern blots containing the same samples were probed with ␣-Rh1 rdgA flies. Parallel blots were probed with ␣-TRP and ␣-tubulin antibodies. antibodies. Size markers (kDa) are indicated. (C) Spatial distribution of gprk1 RNA in adult fly heads. In situ hybridizations to frontal sections were performed with GST Pull-Down Assays. DNAs encoding Drosophila eye-enriched antisense and sense gprk1 probes. A phase-contrast image of the same section G␤ and G␥ were used to prepare the 35S-methionine-labeled hybridized with the gprk1 sense probe is shown. B, brain; M, medulla; L, lamina; R, retina.