key: cord-0005800-kngjiagf authors: Arshavsky, Vadim Yu.; Bownds, M. Deric title: Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP date: 1992 journal: Nature DOI: 10.1038/357416a0 sha: b9eb1963d1f3573180ec7d43971f9d9350e2ef26 doc_id: 5800 cord_uid: kngjiagf THE photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase γ-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP(1–4). In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity(5–7). Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its γ-subunits, accelerates GTPase activity by several fold. Thus the γ-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase α and β-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP. THE photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-actil'ated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase 'Y-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMp l -4. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity5--7. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its 'Y-subunits, accelerates GTPase activity by several fold. Thus the 'Y-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase a and fl-subunits. This cGMP regulation may function in light-adaptation of the photoresponse as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP. We reconstituted photoreceptor membranes which retained transducin but were depleted of cGMP phosphodiesterase (POE), with purified POE or its 'Y·subunits (POE. y )' Photoreceptor membranes containing transducin tightly bound to bleached rhodopsin were obtained bl illuminating the membranes in the absence of nucleotides , More than 80% of the transducin remains bound during multiple hypotonic washes that remove more than 98% of the POE from the membranes, This transducin will turnover if GTP is added: the a-subunit of transducin binds GTP, dissociates from rhodopsin, and slowly hydrolyses GTP to GOP with an intrinsic GTPase activity, It is this GTPase activity that can be altered if the membranes are first permitted to rebind POE, by preincubating them with POE purified from toad rod outer segments to more than 98% homogeneity (Fig. I) , Because the 'Y-subunit of POE is a primary site of POE interaction with transducin 9 , a similar reconstitution with membranes was also done using bovine recombinant POE.,. (ref. 10) , To measure the rate of transducin GTPase activity we started the reaction by adding GTP in an amount less than the transducin present in the sample, so that a single turnover of GTP hydrolysis on each activated transducin molecule occurred. The binding of GTP with transducin under the conditions used here occurs at time zero and the subsequent monitoring of inorganic phosphate (PJ formation reflects the time course of a single synchronized turnover of transducin-bound GTP hydrolysis 11.1 2 . The rate constant of the GTPase reaction can be obtained from an exponential fitting of the data, The rate of the transducin GTPase reaction can be significantly accelerated both by POE and by POE.,. (Fig, 1) , The bottom curve displays the basal GTPase rate of transducin bound to the membranes (0,034 ± 0,003 turnovers per second, n = 6) , Addition of POE or POE.,. accelerates the rate until saturation (0.143±0,02 turnovers per second, n =4 for POE.,. and 0.I4± 0,019 turnovers per second, n = 3 for POE) is observed at -0.3 fJoM POE or -0.6 fJoM POE.,.. Because each POE molecule contains two 'Y-subunits 13, we conclude that the extent of 416 GTPase acceleration in both experiments is determined by the concentration of POE.,.. Figure 2 demonstrates that this acceleration ofGTPase activity is slowed down by cGMP (0,065 ±0,007 turnovers per second; n = 3), and that this slowing requires the presence of the a-and f3-subunits of POE which contain catalytic and non-catalytic cGMP-binding sites I4 -16 • The bottom curve shows that cGMP has no effect on membrane-bound transducin in the absence of POE. It also does not alter the accelerated GTPase rate caused by addition of POE.,. (top curve), It is only if POE is present as the af3'Y2 heterotetramer that cGMP suppresses the accelerated GTPase activity. Two observations 12 suggest that the cGMP-sensitivity is conferred through the non-catalytic binding sites located on the POE aand f3-subunits, The half-saturation of the cGMP effect on transducin GTPase in a suspension of disrupted rod outer seg· ments is very close to the reported value of the-, dissociation constant (Ko) for the POE non-catalytic cGMP-binding sites 14 and -IOO-fold lower than the reported Michaelis constant (K M ) values associated with the catalytic site of amphibian POE t7 • Further, cAMP can bind to and be hydrolysed by the catalytic site, but cannot substitute for cGMP at the site that regulates GTPase. Our findings have several implications for understanding photoreception. It has been a paradox that the rate oftransducin GTPase measured in vitro l8 , 19 is too slow to explain the relatively Photoreceptor membranes (20 fLM rhodopsin) with more than 98% of their PDE extracted but containing -1.6 fLM transducin were preincubated with purified PDE or recombinant PDE.,. at the indicated concentrations. The reaction was started by adding 0.2 fLM [ 'Y-32p]GTP and terminated by perchloric acid. The time course of 'Y-32p, liberation by the GTPase reaction is shown; in all curves 100% hydrolysis corresponds to complete hydrolysis of the 0.2 fLM GTP added. The data for test membranes alone and for the highest concentrations of PDE or PDE.,. are approximated by single exponents. More complicated kinetics are observed for nonsaturating concentrations of POE and POE.,. and the corresponding lines are hand drawn. The data are taken from one of three (8) or four (b) similar experiments. METHODS. The isolation of toad rod outer segments and measurements of single-turnover GTPase activity of transducin was as described in ref. 12 . All experiments were done in buffer A, which contains 100 mM potassium isethionate 10 mM sodium isethionate, 5 mM MgCI 2 , 1 mM dithiothreitol and 15 mM HEPES, pH 7.8. Test membranes retaining >80% of their transducin and depleted by >98% of their PDE were obtained by washing bleached rod outer segments twice with buffer A and twice with buffer B: 5 mM Tris-Hel, pH 8.0,0.5 mM EDTA and 1 mM dithiothreitoL To prepare purified PDE, non-bleached rod outer segments were washed twice with buffer A and three times with 5 mM Tris-Hel, pH 8.0, containing 5 mM MgCI 2 and 1 mM dithiothreitoL Membranes were then bleached to achieve tight binding of most transducin with rhodopsin, and PDE was extracted by buffer B. After concentrating the extract with a Centricon-30 cartridge (Amicon), PDE was purified by a gel-filtration on a Superose-6 column (Pharmacia) equilibrated with buffer B containing 10 mM NaCI. The only detectable impurity in the PDE fraction was -1-2% of transducin's a-subunit which was removed by subsequent chromatography on a protein-A-Superose column (Pharmacia) with immobilized anti-transducin antibodies 4A (ref. 30 ). SDS-polyacrylamide gel electrophoresis of this preparation showed three bands of 92, 87 and 14K corresponding to PDE a-, 13and )I-subunits. Purity was >98%. fast termination of PDE activation observed under similar conditions 20 . More recent work indicates that transducin GTPase can be faster under more physiologial conditions 11,21-23, but the mechanism of GTPase acceleration has remained unclear. The data of Fig. 1 show that PDE itself serves as a GTPase-activating factor. The maximal GTPase rate observed in this reconstitution study (-0.15 S-I) is still about lO-fold slower than the rate of the recovery from a photoresponse. But a more rapid rate (> 0.6 s -I) is observed in suspensions of disrupted rod outer segments l2 for the fast component of GTPase suppressed by micromolar concentrations of cGMP. Our study allows us to conclude that this faster GTPase is a property of that transducin which activates PDE, and thus the extent of PDE-dependent GTPase acceleration is higher in rod outer segment suspensions than in reconstituted membranes. More recent data (V. Y.A. et al., manuscript in preparation) shows that further concentration of rod outer segment suspensions (> 100 fLM rhodopsin) increases GTPase rates by at least twofold, close to the turn-off time of the photoresponse. The data shown in Fig. 2 indicate a feedback mechanism in retinal rods based on cGMP-dependent regulation of the lifetime of activated PDE. Such a mechanism might function during rod background adaptation, when the duration and light sensitivity of the photoresponse is diminished 24 ,25. A reasonable model is that background light depletes intracellular cGMP levels, causing dissociation of cGMP from the non-catalytic binding sites on PDE. This would accelerate the GTPase activity that terminates each PDE activation event, leading to a faster and/or smaller photoresponse. Such a mechanism might work in parallel with the known calcium feedback regulation of adaptation 4 • The regulation of GTP-binding protein GTPase activity by an effector described here, although not previously described for a heterotrimeric G protein, has been documented extensively for several other classes of GTP-binding proteins (for example ref. 26) . It is observed for elongation and initiation factors whose GTPase activity is enhanced by ribosomes. The class of small GTP-binding proteins including the product of proto-oncogene ras interact with GTPase-activating proteins (GAPs) that may also be their effectors 27 . The intrinsic GTPase of the heterotrimeric signal-transducing G proteins is considerably more rapid than that of the small GTP-binding proteins (for example refs 5-7), but still in several systems such as photoreception 2 ,3, 0lfaction 28 and muscarinic receptor-induced potassium channel regulation 29 CORONA VIRUSES, like many animal viruses, are characterized by a restricted host range and tissue tropism 1. Transmissible gastroenteritis virus (TGEV), a major pathogen causing a fatal diarrhoea in newborn pig, replicates selectively in the differentiated enterocytes covering the villi of the small intestine 2 • To investigate the molecular determinants of the infection, we characterized the surface molecule used by the virus for binding and entry into host cells. Here we report that aminopeptidase N, an ectoenzyme abundantly expressed at the apical membrane of the enterocytes, serves as a receptor for TGEV. Monoclonal antibodies were selected for their ability to block infection by TGEV of porcine cell lines. They recognized a brush-border membrane protein of Mr lSOK, which was identified as aminopeptidase N by amino-terminal sequencing. Two lines of evidence supported the view that the peptidase itself :j: To whom correspondence should be addressed. 1 gen Gray·Keller for participating in the preliminary stage of this work