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• W2000444382 abstract "Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nm. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ∼10 μm [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (∼110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs. Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nm. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ∼10 μm [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (∼110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs. In outer segments of vertebrate retinal photoreceptors, rhodopsin absorbs a photon which in turn triggers GTP-dependent activation of cGMP phosphodiesterase. The activated phosphodiesterase hydrolyzes cGMP. The resulting decrease in cytoplasmic [cGMP] leads to reduction in the activity of cGMP-gated cation channels and hyperpolarization of plasma membranes (1Hurley J.B. Annu. Rev. Physiol. 1987; 49: 793-812Crossref PubMed Google Scholar, 2Yau K.-W. Baylor D.A. Annu. Rev. Neurosci. 1989; 12: 289-327Crossref PubMed Scopus (436) Google Scholar, 3Pugh Jr., E.N. Lamb T.D. Biochim. Biophys. Acta. 1993; 1141: 111-149Crossref PubMed Scopus (522) Google Scholar, 4Koutalos Y. Yau K.-W. Trends Neurosci. 1996; 19: 73-81Abstract Full Text PDF PubMed Scopus (216) Google Scholar). Restoration of the dark membrane potential requires recovery of the dark level of cytoplasmic [cGMP]. Therefore, GC, 1The abbreviations used are: GC, guanylyl cyclase; retGC, retinal guanylyl cyclase; GCAP, guanylyl cyclase-activating protein; ROS, rod outer segments; GCAP-1m, a constitutively active mutant of GCAP-1, Y99C; GCAP-2m, a constitutively active mutant of GCAP-2, E80Q/E160Q/D158N; BS3, bis(sulfosuccinimidyl) suberate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride the enzyme that converts GTP to cGMP, has a crucial role in visual transduction. A retinal membrane GC has been purified from frog, toad, and bovine photoreceptor outer segments (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar, 6Koch K.-W. J. Biol. Chem. 1991; 266: 8634-8637Abstract Full Text PDF PubMed Google Scholar) and identified as a ∼110 kDa protein. Isozymes of the GC have also been shown biochemically (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar, 7Hakki S. Sitaramayya A. Biochemistry. 1990; 29: 1088-1094Crossref PubMed Scopus (47) Google Scholar). Subsequently two forms of membrane GC (retGC-1, ROS-GC1 or GC-E, and retGC-2, ROS-GC2 or GC-F) were cloned from human, bovine, and rat retinal cDNA libraries (8Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.U. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 9Goraczniak R.M. Duda T. Sitaramayya A. Sharma R.K. Biochem. J. 1994; 302: 455-461Crossref PubMed Scopus (129) Google Scholar, 10Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (239) Google Scholar, 11Yang R.B. Foster D.C. Garbers D.L. Fiille H.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (219) Google Scholar, 12Goraczniak R.M. Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1997; 234: 666-670Crossref PubMed Scopus (52) Google Scholar). The structure of these retGCs indicates that the enzyme is a member of the peptide-regulated, membrane-bound GC family, although the retGC is not activated by known peptides. Four functional domains in retGCs have been predicted: a N-terminal extracellular domain, a transmembrane domain, an intercellular protein-kinase-like domain, and a C-terminal catalytic domain. Immunocytochemistry has shown that retGC-1 is localized primarily in cone outer segments and to a lesser extent in rod outer segments (13Liu X. Seno k. Nishizawa Y. Hayashi F. Yamazaki A. Matsumoto H. Wakabayashi T. Usukura J. Exp. Eye Res. 1994; 59: 761-768Crossref PubMed Scopus (127) Google Scholar, 14Dizhoor A.M. Lowe D.G. Olshevskaya E.V. Laura R.P. Hurley J.B. Neuron. 1994; 12: 1345-1352Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 15Cooper N. Liu L. Yoshida A. Pozdnyakov N. Margulis A. Sitaramayya A. J. Mol. Neurosci. 1996; 6: 211-222Crossref Scopus (47) Google Scholar). RetGC-1 is also detected in the plexiform layers of retina (13Liu X. Seno k. Nishizawa Y. Hayashi F. Yamazaki A. Matsumoto H. Wakabayashi T. Usukura J. Exp. Eye Res. 1994; 59: 761-768Crossref PubMed Scopus (127) Google Scholar, 15Cooper N. Liu L. Yoshida A. Pozdnyakov N. Margulis A. Sitaramayya A. J. Mol. Neurosci. 1996; 6: 211-222Crossref Scopus (47) Google Scholar), leading to speculation that the enzyme is not unique in photoreceptor outer segments. RetGC-2 is in photoreceptors (10Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (239) Google Scholar); however, detailed localization of the enzyme in the retina has not been demonstrated. RetGC-1 appears to be more abundant than retGC-2 in the retina (10Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (239) Google Scholar), and only retGC-1 may contribute to the pool of cGMP essential to support phototransduction in cone photoreceptors (16Semple-Rowland S.L. Lee N.R. van Hooser J.P. Palczewski K. Baehr W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1271-1276Crossref PubMed Scopus (130) Google Scholar). The reduction of cGMP-gated channel activity by lowering cytoplasmic [cGMP] blocks Na2+ and Ca2+ influx, and allows a Na2+/Ca2+, K+ exchanger to decrease cytoplasmic [Ca2+] (4Koutalos Y. Yau K.-W. Trends Neurosci. 1996; 19: 73-81Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 17Gray-Keller M.P. Detwiler P.B. Neuron. 1994; 13: 849-861Abstract Full Text PDF PubMed Scopus (236) Google Scholar). When [Ca2+] is low, retGC is stimulated (18Koch K.-W. Stryler L. Nature. 1988; 334: 64-66Crossref PubMed Scopus (473) Google Scholar). In contrast to other membrane-bound GCs that are regulated by binding of peptides to their extracellular domain (19Garbers D.L. J. Biol. Chem. 1989; 264: 9103-9106Abstract Full Text PDF PubMed Google Scholar, 20Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar), this Ca2+-sensitive stimulation of retGC is mediated by at least two calmodulin-like Ca2+-binding proteins termed GCAPs, 1 and 2 (14Dizhoor A.M. Lowe D.G. Olshevskaya E.V. Laura R.P. Hurley J.B. Neuron. 1994; 12: 1345-1352Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 21Gorczyca W.A. Grey-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Crossref PubMed Scopus (212) Google Scholar, 22Palczewski K. Subbaraya I. Gorczyca W.A. Hebkar B.S. Ruiz C.C. Ohguro H. Huang T. Zhao X. Crabb J.W. Johnson R.S. Walsh K.A. Gray-Keller M.P. Detwiler P.B. Baehr W. Neuron. 1994; 13: 395-404Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 23Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankouclinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). GCAPs interact with the intracellular domain of the enzyme (24Duda T. Goraczniak R. Surgucheva I. Rudnicka-Nawrot M. Gorczyca W.A. Palczewski K. Sitaramayya A. Baehr W. Sharma R.K. Biochemistry. 1996; 35: 8478-8482Crossref PubMed Scopus (114) Google Scholar, 25Laura R.P. Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 11646-11651Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). When free [Ca2+] is less than ∼300 nm, retGC-1 is activated by GCAPs. RetGC-2 has also been reported to be stimulated by GCAP-2 under similar [Ca2+] (10Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (239) Google Scholar, 12Goraczniak R.M. Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1997; 234: 666-670Crossref PubMed Scopus (52) Google Scholar, 26Goraczniak R.M. Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1998; 245: 447-453Crossref PubMed Scopus (34) Google Scholar). In addition, GCAPs appear to inhibit the basal GC activity in the presence of the higher [Ca2+] (more than ∼500 nm) (27Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 19346-19350Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Dizhoor A.M. Borkov S.G. Olshevskaya E.V. J. Biol. Chem. 1998; 273: 17311-17314Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Thus, the regulatory mechanism of retGC is completely different from that of peptide-regulated GCs. GCAP-1 is mainly detected in cone outer segments, in particular, in disc membrane regions (29Howes K. Bronson J.D. Dang Y.L. Li N. Zhang K. Ruiz C. Helekar B. Lee M. Subbaraya I. Chen J. Baehr W. Invest. Ophthalmol. Vis. Sci. 1998; 39: 867-875PubMed Google Scholar, 30Cuenca N. Lopez S. Howes K. Kolb H. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1243-1250PubMed Google Scholar, 31Kachi S. Nishizawa Y. Olshevskaya E.V. Yamazaki A. Miyake Y. Dizhoor A.M. Usukura J. Exp. Eye Res. 1999; 68: 465-473Crossref PubMed Scopus (67) Google Scholar). GCAP-1 is also observed in rod outer segments, but the content is much lower than that in cone outer segments. Less GCAP-1 is also found in synaptic regions and inner segments of cones. GCAP-2 is predominantly observed in outer and inner segments of rods and cones (23Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankouclinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 29Howes K. Bronson J.D. Dang Y.L. Li N. Zhang K. Ruiz C. Helekar B. Lee M. Subbaraya I. Chen J. Baehr W. Invest. Ophthalmol. Vis. Sci. 1998; 39: 867-875PubMed Google Scholar, 30Cuenca N. Lopez S. Howes K. Kolb H. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1243-1250PubMed Google Scholar, 31Kachi S. Nishizawa Y. Olshevskaya E.V. Yamazaki A. Miyake Y. Dizhoor A.M. Usukura J. Exp. Eye Res. 1999; 68: 465-473Crossref PubMed Scopus (67) Google Scholar). Synaptic regions are also labeled by a GCAP-2 antibody. It has also been reported that Ca2+-binding proteins of the S100 family, especially S100b, activate retGC-1 in the presence of high [Ca2+] (32Pozdnyakov N. Yoshida A. Cooper N.G.F. Margulis A. Duda T. Sharma R.K. Sitaramayya A. Biochemistry. 1995; 34: 14279-14283Crossref PubMed Scopus (60) Google Scholar, 33Margulis A. Pozdnyakov N. Sitaramayya A. Biochem. Biophys. Res. Commun. 1995; 218: 243-247Crossref Scopus (64) Google Scholar). Half-maximal activation was observed at about 40∼50 μm [Ca2+] (33Margulis A. Pozdnyakov N. Sitaramayya A. Biochem. Biophys. Res. Commun. 1995; 218: 243-247Crossref Scopus (64) Google Scholar). Therefore, it is believed that S100 proteins are not involved in phototransduction. Additional mechanisms and factors may also be involved in the regulation of retGCs, including phosphorylation (34Wolbring G. Schnetkamp P.P. Biochemistry. 1996; 35: 11013-11018Crossref PubMed Scopus (20) Google Scholar, 35Aparicio J.B. Applebury M.L. J. Biol. Chem. 1996; 271: 27083-27089Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), ATP binding (36Sitaramayya A. Marala R.B. Hakki S. Sharma R.K. Biochemistry. 1991; 30: 6742-6747Crossref PubMed Scopus (21) Google Scholar, 37Gorczyca W.A. van Hooser J.P. Palczewski K. Biochemistry. 1994; 33: 3217-3222Crossref PubMed Scopus (51) Google Scholar, 38Tucker C.L. Laura R.P. Hurley J.B. Biochemistry. 1997; 36: 11995-12000Crossref PubMed Scopus (31) Google Scholar), actin binding (39Hallett M.A. Delaat J.L. Arikawa K. Schlamp C.L. Kong F.S. Williams D.S. J. Cell Sci. 1996; 109: 18003-18012Google Scholar), an inhibitor (guanylyl cyclase-inhibitory protein) of GCAP-activated retGC in amphibian retina (40Li N. Fariss R.N. Zhang K. Otto-Buco A. Haeseleer F. Bronson D. Qin N. Yamazaki A. Subbaraya I. Milam A.H. Palczewski K. Baehr W. Eur. J. Biochem. 1998; 252: 591-599Crossref PubMed Scopus (44) Google Scholar), and inhibition of retGC-1 by RGS9 (41Seno K. Kishizawa A. Ihara S. Maeda T. Bondarenko V.A. Nishizawa Y. Usukura J. Yamazaki A. Hayashi F. J. Biol. Chem. 1998; 273: 22169-22172Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Characterizations of membrane-bound GC and adenylyl cyclase have suggested that the mechanisms for the expression of these enzymatic activities are closely related (42Tucker C.L. Hurley J.H. Miller T.R. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5993-5997Crossref PubMed Scopus (187) Google Scholar, 43Sunahara R.K. Beuve A. Tesmer J.J.G. Sprang S.R. Garbers D.L. Gilman A.G. J. Biol. Chem. 1998; 273: 16332-16338Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and that at least two cyclase catalytic consensus domains may be required for the activity of these cyclases. Membrane adenylyl cyclase contains two putative catalytic domains that when separately expressed have no activity (44Tang W. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar). Peptide-regulated GCs have also been proposed to exist as dimeric or oligomeric forms even in the absence of ligands (45Iwata T. Uchida-Mizuno K. Katafuchi T. Ito T. Hagiwara H. Hirose S. J. Biochem. (Tokyo). 1991; 110: 73-79Crossref Scopus (50) Google Scholar, 46Lowe D.G. Biochemistry. 1992; 31: 10421-10425Crossref PubMed Scopus (75) Google Scholar, 47Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (153) Google Scholar). A recent study has also suggested that retGCs form homodimers in photoreceptor outer segments (48Yang R.-B. Garbers D.L. J. Biol. Chem. 1997; 272: 13738-13742Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). However, it remains unknown whether dimerization or oligomerization of retGCs is related to the regulation of retGC activity. This question is important especially because the activity of retGCs is regulated so differently from peptide-regulated GCs and the regulatory mechanism of retGCs by GCAPs may not be identical to that of peptide-regulated GCs. Thus, we investigated the relationship between activation of retGC-1 by GCAPs and dimerization of the enzyme. In addition to GCAPs, we used constitutively active mutants of GCAPs and S100b to show that dimerization of retGC-1 is related to its activation, not to Ca2+-binding proteins or [Ca2+]. Dimerization of retGC-1 was monitored using a cross-linker under the conditions similar to those used for the measurement of the GC activity. Our results suggest that GCAPs activate retGC-1 by enhancing its dimerization, and that transition of the active form of retGC-1 to its inactive form is caused by either partial or complete dissociation of the dimeric form. Dark-adapted frozen bovine retinas were purchased from Dr. Yee-Kin Ho (University of Illinois, Department of Biochemistry, Chicago). Other materials were purchased from the following sources: Sephacryl S-200 HR from Pharmacia Biotech Inc.; [α-32P]GTP and [3H]cGMP from NEN Life Science Products Inc.; cGMP and GTP from Roche Molecular Biochemicals; AG 1-X2 resin from Bio-Rad; alumina N-Super I from ICN; creatine phosphokinase, phosphocreatine, phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, 1-methyl-3-isobutylxanthine,n-dodecyl-β-d-maltoside, S100b, GTP-agarose resin, and high molecular weight makers for SDS-PAGE from Sigma; Ultra Super Signal substrate, PVDF membranes, BS3, 3,3′-dithiobis(sulfosuccinimidyl propionate), and disuccinimidyl suberate from Pierce. Antibodies against retGC-1 (13Liu X. Seno k. Nishizawa Y. Hayashi F. Yamazaki A. Matsumoto H. Wakabayashi T. Usukura J. Exp. Eye Res. 1994; 59: 761-768Crossref PubMed Scopus (127) Google Scholar), GCAPs (23Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankouclinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 31Kachi S. Nishizawa Y. Olshevskaya E.V. Yamazaki A. Miyake Y. Dizhoor A.M. Usukura J. Exp. Eye Res. 1999; 68: 465-473Crossref PubMed Scopus (67) Google Scholar), and RGS9 (41Seno K. Kishizawa A. Ihara S. Maeda T. Bondarenko V.A. Nishizawa Y. Usukura J. Yamazaki A. Hayashi F. J. Biol. Chem. 1998; 273: 22169-22172Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) were prepared as described. Bovine ROS were prepared from dark-adapted frozen retinas as described (49Yamazaki A. Tatsumi M. Bitensky M.W. Methods Enzymol. 1988; 159: 702-710Crossref PubMed Scopus (39) Google Scholar). Bleached ROS membranes from 20 retinas were suspended in 3 ml of Buffer A (10 mm HEPES (pH 7.5), 5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 5 μmleupeptin, 5 μm pepstatin A, and 100 μmCaCl2), homogenized by passing through a No. 21 needle 10 times and centrifuged (200,000 × g, 4 °C, 15 min) (x 7). The membranes were further washed (3 times) with Buffer B (10 mm HEPES (pH 7.5), 5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluorine, 5 μmleupeptin, and 5 μm pepstatin A), suspended in 3 ml of Buffer B, frozen with liquid nitrogen, and stored at −80 °C. The washed membranes are termed GCAPs-free ROS membranes. Purified retGC from bovine ROS was prepared using a GTP-agarose column (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar). The purity of the preparation was greater than 95%. The purified retGC was stored in liquid nitrogen until used (up to months). Preparation of recombinant bovine retGC-1 expressed in COS-7 cells (24Duda T. Goraczniak R. Surgucheva I. Rudnicka-Nawrot M. Gorczyca W.A. Palczewski K. Sitaramayya A. Baehr W. Sharma R.K. Biochemistry. 1996; 35: 8478-8482Crossref PubMed Scopus (114) Google Scholar), bovine GCAPs in Escherichia coli (28Dizhoor A.M. Borkov S.G. Olshevskaya E.V. J. Biol. Chem. 1998; 273: 17311-17314Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 50Olshevskaya E.V Hughes R.E. Hurley J.B. Dizhoor A.M. J. Biol. Chem. 1997; 272: 14327-14333Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and constitutively active mutants of GCAP-1 (Y99C) (28Dizhoor A.M. Borkov S.G. Olshevskaya E.V. J. Biol. Chem. 1998; 273: 17311-17314Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and GCAP-2 (E80Q/E160Q/D158N) (27Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 19346-19350Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) has been described. GC activity was measured as described (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar). Our preliminary studies indicated that 5 μg of protein of a ROS homogenate had high GC activity with negligible hydrolysis of cGMP under assay conditions. Thus, 5 μg of protein of ROS homogenate or GCAPs-free ROS membranes were used for all studies. Briefly, ROS membranes (5 μg of protein) were incubated with 200 μl of Buffer C (50 mm HEPES (pH 7.5), 1 mm GTP, 1 mm cGMP, 2 mm 1-methyl-3-isobutylxanthine, 5 mm MgCl2, 15 mm phosphocreatine, 50 μg/ml creatine phosphokinase, ∼5 μCi of [α-32P]GTP, and ∼0.5 μCi of [3H]cGMP). The reaction was initiated by addition of GTP and cGMP. Following incubation (37 °C for 10 min), the reaction was terminated by adding 40 μl of 1 n HCl and boiling for 2 min. [32P]cGMP derived from [α-32P]GTP was isolated by alumina and AG 1-X2 columns (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar), and both3H and 32P radioactivities in samples were counted. In order to obtain the exact relationship between dimerization of retGC-1 and its activity, cross-linking reaction of retGC-1 was carried out under conditions similar to that for the measurement of GC activity, although the protein concentrations used were different because each experiment was performed in its linear range. A bovine ROS homogenate, GCAPs-free ROS membranes or COS-7 cell membranes containing recombinant bovine retGC-1 were used. The preparation was suspended in 50 μl of Buffer D (50 mm HEPES (pH 7.5), 1 mm GTP, 1 mm cGMP, and 5 mm MgCl2). Pilot experiments indicated that the optimal conditions for the cross-linking reaction by BS3 were the following: BS3concentration, 50 μm (Fig. 1); temperature for the cross-linking reaction, 0 °C; incubation period for the cross-linking reaction, 30 min; and protein, 50 μg. We note that the GTP regeneration system and proteinase inhibitors added in the GC assay mixture did not affect the cross-linking reactions. We also note that the presence of 1 mm GTP in the cross-linking reaction mixture slightly (less than 10%) inhibited the formation of the 210-kDa cross-linked product, but 1 mm cGMP did not. The cross-linking reaction was terminated by addition of SDS-sample buffer and boiling for 5 min. The cross-linked products were immediately separated by SDS-PAGE (5–20% acrylamide gradient). After electrophoresis, proteins were blotted to PVDF membranes and cross-linked products of retGC-1 were detected with a retGC-1-specific antibody (13Liu X. Seno k. Nishizawa Y. Hayashi F. Yamazaki A. Matsumoto H. Wakabayashi T. Usukura J. Exp. Eye Res. 1994; 59: 761-768Crossref PubMed Scopus (127) Google Scholar) and chemiluminescent autoradiography using ULTRA Super Signal substrate. The bands of cross-linked products were scanned by Paragon 1200A3 Pro Scanner and the relative density (mm2 × OD) was calculated by Molecular Analyst Software (Bio-Rad). It should be emphasized that the R f value of the 210-kDa cross-linked product was slightly different in each SDS-PAGE; however, the molecular mass of the 210-kDa product was constantly monitored by myosin (205 kDa) as a molecular standard. The molecular mass of the >400-kDa cross-linked product(s) was estimated using high molecular mass (97,400–584,400) standards. The freshly solubilized retGC was prepared by incubation of ROS membranes with 1 ml of Buffer E (20 mm HEPES (pH 7.5), 1 mmdithiothreitol, 5 mm MgCl2, 0.1 mmphenylmethylsulfonyl fluoride, 5 μm leupeptin, 5 μm pepstatin A, and 150 mm NaCl) containing 5% n-dodecyl-β-d-maltoside for 2 h at 0 °C (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar). After centrifugation (100,000 × g, 4 °C, 30 min), a portion of the supernatant (1.5 mg of protein, 200 μl) was immediately applied to a Sephacryl S-200 HR column (9 × 600 mm) which had been equilibrated with Buffer E containing 0.1%n-dodecyl-β-d-maltoside. Another portion of the supernatant was stored on ice overnight, and applied to the column under the same conditions. Purified retGC (8 μg) was also applied to the column under the same conditions. Column chromatography conditions were as follows: flow rate, 1.5 ml/10 min; and fraction volume, 1.0 ml. For column calibration, proteins with known Stokes radii were chromatographed under identical conditions. SDS-PAGE was performed as described (51Yamazaki A. Tatsumi M. Torney D.C. Bitensky M.W. J. Biol. Chem. 1987; 262: 9316-9323Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were assayed with bovine serum albumin as standard (52Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Protein concentration in samples containing a detergent was monitored as described (5Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (80) Google Scholar). Ca-EGTA buffers were calculated (26Goraczniak R.M. Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1998; 245: 447-453Crossref PubMed Scopus (34) Google Scholar) and prepared (53Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (399) Google Scholar). Free [Ca2+] was verified by Ca2+-electrode and Rod-2 titration. It should be emphasized that all experiments were carried out more than two times, and the results were similar. Data shown are representative of these experiments. Dimerization or oligomerization of peptide-regulated GCs have been monitored using several methods including cross-linking (45Iwata T. Uchida-Mizuno K. Katafuchi T. Ito T. Hagiwara H. Hirose S. J. Biochem. (Tokyo). 1991; 110: 73-79Crossref Scopus (50) Google Scholar), measurement of molecular weight (46Lowe D.G. Biochemistry. 1992; 31: 10421-10425Crossref PubMed Scopus (75) Google Scholar), and the yeast two-hybrid system (47Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (153) Google Scholar). In this study, to obtain the exact relationship between dimerization of retGC-1 and its activity, we attempted to fix dimeric (or oligomeric) forms of retGC-1 by cross-linking under a condition similar to these used for measurement of GC activity. We found that two oligomeric forms of the retGC-1, ∼210 and >400 kDa, were fixed by less than 60 μm BS3 in a ROS homogenate (Fig.1, upper inset). The amounts of both cross-linked products were increased in a [BS3]-dependent manner; however, the 210-kDa product was detected by a much lower [BS3] than the >400-kDa product. These cross-linked products in ROS homogenates were also formed with different cross-linkers, such as 3,3′-dithiobis(sulfosuccinimidyl propionate) and disuccinimidyl suberate. These two cross-linked products of retGC-1 were also observed in a purified retGC preparation (Fig. 1, lower inset), indicating that these oligomers are made from retGC. Because the molecular mass (∼210 kDa) of the cross-linking product is similar to the calculated molecular mass of the retGC-1 dimer (∼220 kDa), we conclude that the 210-kDa product is a dimer of retGC-1. We note that the 210-kDa cross-linked product was detected in ROS homogenates only when [Ca2+] was low (data not shown), and that the amount of 210-kDa product in the purified retGC-1 preparation was much less than that in ROS homogenates. RetGC-1 has been shown to be regulated by proteins, such as GCAPs (14Dizhoor A.M. Lowe D.G. Olshevskaya E.V. Laura R.P. Hurley J.B. Neuron. 1994; 12: 1345-1352Abstract Full Text PDF PubMed Scopus (273) Google Scholar,21Gorczyca W.A. Grey-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Crossref PubMed Scopus (212) Google Scholar, 22Palczewski K. Subbaraya I. Gorczyca W.A. Hebkar B.S. Ruiz C.C. Ohguro H. Huang T. Zhao X. Crabb J.W. Johnson R.S. Walsh K.A. Gray-Keller M.P. Detwiler P.B. Baehr W. Neuron. 1994; 13: 395-404Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 23Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankouclinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full" @default.
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• W2000444382 title "Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization" @default.
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• W2000444382 cites W1514044463 @default.
• W2000444382 cites W1583253515 @default.
• W2000444382 cites W1599050967 @default.
• W2000444382 cites W1600119412 @default.
• W2000444382 cites W1941649185 @default.
• W2000444382 cites W1952786888 @default.
• W2000444382 cites W1969609232 @default.
• W2000444382 cites W1971338242 @default.
• W2000444382 cites W1972471590 @default.
• W2000444382 cites W1977571897 @default.
• W2000444382 cites W1979386817 @default.
• W2000444382 cites W1983094993 @default.
• W2000444382 cites W1984323462 @default.
• W2000444382 cites W1987029335 @default.
• W2000444382 cites W1988127541 @default.
• W2000444382 cites W1989352995 @default.
• W2000444382 cites W1989566939 @default.
• W2000444382 cites W1989678684 @default.
• W2000444382 cites W1992575517 @default.
• W2000444382 cites W2004660422 @default.
• W2000444382 cites W2010730039 @default.
• W2000444382 cites W2019615226 @default.
• W2000444382 cites W2021026506 @default.
• W2000444382 cites W2021100480 @default.
• W2000444382 cites W2024664315 @default.
• W2000444382 cites W2028274336 @default.
• W2000444382 cites W2037854344 @default.
• W2000444382 cites W2040943511 @default.
• W2000444382 cites W2045477387 @default.
• W2000444382 cites W2048965038 @default.
• W2000444382 cites W2049574152 @default.
• W2000444382 cites W2062372752 @default.
• W2000444382 cites W2065110896 @default.
• W2000444382 cites W2068693331 @default.
• W2000444382 cites W2070237221 @default.
• W2000444382 cites W2073513922 @default.
• W2000444382 cites W2079690597 @default.
• W2000444382 cites W2081311184 @default.
• W2000444382 cites W2085556812 @default.
• W2000444382 cites W2085894867 @default.
• W2000444382 cites W2087280841 @default.
• W2000444382 cites W2089906989 @default.
• W2000444382 cites W2090897867 @default.
• W2000444382 cites W2105129317 @default.
• W2000444382 cites W2113585002 @default.
• W2000444382 cites W2156028270 @default.
• W2000444382 cites W2169776140 @default.
• W2000444382 cites W2176431604 @default.
• W2000444382 cites W2181543902 @default.
• W2000444382 cites W2221521962 @default.
• W2000444382 cites W2527494488 @default.
• W2000444382 cites W4293247451 @default.
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