Instead of Binding Calcium, One of the EF-hand Structures in Guanylyl Cyclase Activating Protein-2 Is Required for Targeting Photoreceptor Guanylyl Cyclase
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m107539200
ISSN1083-351X
AutoresAlexandre N. Ermilov, Elena V. Olshevskaya, Alexander M. Dizhoor,
Tópico(s)Photochromic and Fluorescence Chemistry
ResumoGuanylyl cyclaseactivator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca2+-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca2+ concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca2+ and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca2+-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca2+-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca2+-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca2+ for the ability to interact with the target enzyme. Guanylyl cyclaseactivator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca2+-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca2+ concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca2+ and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca2+-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca2+-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca2+-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca2+ for the ability to interact with the target enzyme. photoreceptor membrane guanylyl cyclase guanylyl cyclase activating protein polymerase chain reaction 4-morpholinepropanesulfonic acid Light-induced hyperpolarization of the vertebrate photoreceptor plasma membrane inhibits the release of the neuromediator, glutamate, from the synaptic termini of rods and cones and thus generates the signal for the secondary neurons of the retina. As the first step in visual signal transduction, photoisomerized rhodopsin triggers hydrolysis of cGMP by activating a G-protein, transducin, that subsequently stimulates a cGMP phosphodiesterase, PDE6, and this causes cGMP-gated cation channels to close (see Refs. 1Baylor D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 560-565Crossref PubMed Scopus (273) Google Scholar, 2Pugh Jr., E.N. Duda T. Sitaramayya A. Sharma R.K. Biosci. Rep. 1997; 17: 429-473Crossref PubMed Scopus (156) Google Scholar, 3Pugh Jr., E.N. Lamb T.D. Biochim. Biophys. Acta. 1993; 1141: 111-149Crossref PubMed Scopus (512) Google Scholar for review). Both rods and cones can quickly recover to their resting potential after the excitation induced by a non-saturating flash of light. Exposure of photoreceptors to a constant illumination at first saturates their response, but through a complex process of light adaptation, the cells can reopen cGMP-gated cation channels and partially restore their light sensitivity. Ca2+-sensitive synthesis of cGMP by membrane guanylyl cyclase (retGC)1plays one of the major roles among multiple reactions that result in reopening of the cGMP-gated channels during recovery and light adaptation (4Koch K.W. Stryer L. Nature. 1988; 334: 64-66Crossref PubMed Scopus (468) Google Scholar, 5Pugh Jr., E.N. Nikonov S. Lamb T.D. Curr. Opin. Neurobiol. 1999; 9: 410-418Crossref PubMed Scopus (268) Google Scholar). A Na+/Ca2+,K+-exchanger continuously extrudes Ca2+ ions from the photoreceptor outer segment; therefore, when cGMP is hydrolyzed and the Na+/Ca2+ influx is stopped, the intracellular Ca2+ concentrations in rods and cones can decrease from near 500–600 nm in the dark down to 50 nm in the light (6Gray-Keller M.P. Detwiler P.B. Neuron. 1994; 13: 849-861Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 7Sampath A.P. Matthews H.R. Cornwall M.C. Bandarchi J. Fain G.L. J. Gen. Physiol. 1999; 113: 267-277Crossref PubMed Scopus (90) Google Scholar). In response to the decrease in free Ca2+concentrations, Ca2+-binding proteins, GCAPs (reviewed in Refs. 8Dizhoor A.M. Hurley J.B. Methods. 1999; 19: 521-531Crossref PubMed Scopus (65) Google Scholar, 9Dizhoor A.M. Cell Signal. 2000; 12: 711-719Crossref PubMed Scopus (51) Google Scholar, 10Palczewski K. Polans A.S. Baehr W. Ames J.B. Bioessays. 2000; 22: 337-350Crossref PubMed Scopus (134) Google Scholar), accelerate synthesis of cGMP by retGC (reviewed in Refs.11Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar and 12Garbers D.L. Methods. 1999; 19: 477-484Crossref PubMed Scopus (53) Google Scholar).Two homologous GCAPs (GCAP-1 and -2) have been directly isolated from the retina, and the existence of a gene for the third homologue, GCAP-3, has been revealed by cDNA cloning in some vertebrate species (13Gorczyca W.A. Gray-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Crossref PubMed Scopus (212) 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 (270) Google Scholar, 15Haeseleer F. Sokal I. Li N. Pettenati M. Rao N. Bronson D. Wechter R. Baehr W. Palczewski K. J. Biol. Chem. 1999; 274: 6526-6535Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). GCAP-2 is highly expressed in rods, whereas GCAP-1 is expressed at high levels in cones and at lower levels in rods (16Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 17Howes K. Bronson J.D. Dang Y.L. Li N. Zhang K. Ruiz C. Helekar B. Lee M. Subbaraya I. Kolb H. Chen J. Baehr W. Invest. Ophthalmol. Vis. Sci. 1998; 39: 867-875PubMed Google Scholar, 18Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Crossref PubMed Scopus (82) Google Scholar, 19Kachi S. Nishizawa Y. Olshevskaya E. Yamazaki A. Miyake Y. Wakabayashi T. Dizhoor A. Usukura J. Exp. Eye Res. 1999; 68: 465-473Crossref PubMed Scopus (65) Google Scholar). Disruption of both GCAP-1 and GCAP-2 genes results in abnormally slow recovery in mouse rods, especially in response to strong flash of light, consistently with the expected slower accumulation of cGMP in the absence of GCAPs. Although the relative contribution of GCAP-1 and -2 to the kinetics of dim flash responses in rods and cones remains unclear, GCAP-2 in vitro stimulates the activity of both known isozymes of retGC (retGC-1 and -2) present in photoreceptor membranes (20Lowe 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 (234) Google Scholar), and expression of GCAP-2 alone in GCAP-1/GCAP-2 knockout mice can restore the rate at which rods recover after a bright flash of light (21Mendez A. Burns M.E. Sokal I. Dizhoor A.M. Baehr W. Palczewski K. Baylor D.A. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9948-9953Crossref PubMed Scopus (210) Google Scholar).GCAPs are closely related to other recoverin-like proteins (22Nef P. Guidebook to the Calcium-binding Proteins.in: Celio M.R. Pauls T.L. Shwaller B. Oxford University Press, New York1996: 15-20Google Scholar, 23Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar) within the EF-hand superfamily. Similar to other members of this family, GCAPs are 24-kDa N-fatty-acylated proteins that contain four helix-turn-helix EF-hand structures (EF-1 through EF-4, Fig. 1), of which three (EF-2, -3, and -4) determine Ca2+sensitivity of GCAPs. Similarly to other proteins of this group, in the first EF-hand domain, EF-1, the amino acid sequence corresponding to the Ca2+-binding loop is disrupted and does not have all of the proper side chain residues required for binding Ca2+ion (22Nef P. Guidebook to the Calcium-binding Proteins.in: Celio M.R. Pauls T.L. Shwaller B. Oxford University Press, New York1996: 15-20Google Scholar, 23Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). In their Ca2+-free form GCAPs stimulate the activity of guanylyl cyclase, but upon binding Ca2+ they undergo an activator-to-inhibitor transition (24Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 19346-19350Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 25Rudnicka-Nawrot M. Surgucheva I. Hulmes J.D. Haeseleer F. Sokal I. Crabb J.W. Baehr W. Palczewski K. Biochemistry. 1998; 37: 248-257Crossref PubMed Scopus (84) Google Scholar).GCAPs activate retGC by enhancing dimerization of the cyclase subunits (26Yu H. Olshevskaya E. Duda T. Seno K. Hayashi F. Sharma R.K. Dizhoor A.M. Yamazaki A. J. Biol. Chem. 1999; 274: 15547-15555Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) required for the cyclase catalytic activity (27Ramamurthy V. Tucker C. Wilkie S.E. Daggett V. Hunt D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 26218-26229Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Ca2+-free GCAP-2 can itself form a stable homodimer that can be detected by high resolution gel chromatography (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Its dimerization is highly Ca2+-sensitive so that Ca2+-loaded GCAP-2 quickly dissociates into monomers (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Previous results indicate that dimerization of the Ca2+-free GCAP-2 is likely to be a part of a mechanism ("dimer-adapter" hypothesis) by which it modulates the interaction between the cyclase subunits (9Dizhoor A.M. Cell Signal. 2000; 12: 711-719Crossref PubMed Scopus (51) Google Scholar, 28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).The three-dimensional structure of Ca2+-loaded GCAP-2 is very similar to that of recoverin and neurocalcin (29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), although the structure of the Ca2+-free (activator) form of GCAP-2 remains undetermined. Potential binding sites for GCAP-2 in retGC have been studied using synthetic peptides, chemical cross-linking, and deletion analyses. According to these studies, there are several fragments in retGC kinase homology and catalytic domains that can make contact with GCAPs (30Sokal I. Haeseleer F. Arendt A. Adman E.T. Hargrave P.A. Palczewski K. Biochemistry. 1999; 38: 1387-1393Crossref PubMed Scopus (48) Google Scholar, 31Lange C. Duda T. Beyermann M. Sharma R.K. Koch K.W. FEBS Lett. 1999; 460: 27-31Crossref PubMed Scopus (66) Google Scholar, 32Krylov D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 30648-30654Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, the location of the binding sites for retGC within the GCAP-2 molecule remains rather obscure. Previous efforts to map functionally significant regions in GCAP-2 using GCAP-2/neurocalcin and GCAP-2/recoverin chimeras or deletion mutants revealed that in addition to the three Ca2+-binding EF-hand loops, there were three segments of the molecule that could not be exchanged for the corresponding regions from other recoverin-like proteins without loss of GCAP function as a Ca2+-sensitive cyclase regulator (33Olshevskaya E.V. Boikov S. Ermilov A. Krylov D. Hurley J.B. Dizhoor A.M. J. Biol. Chem. 1999; 274: 10823-10832Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Surprisingly, one of those three regions, Lys29–Phe48, included the first EF-hand-like motif that lost its ability to bind Ca2+. In the present paper, we demonstrate that various point mutations in this region can specifically inhibit interactions of GCAP-2 with the target enzyme. All tested EF-1 mutants of GCAP-2 that were unable to stimulate or inhibit the cyclase were still able to form dimers in the absence of Ca2+, similar to the wild type GCAP-2. We conclude that the first EF-hand-like motif in GCAP-2 participates in targeting retGC. These results are consistent with the model according to which GCAP-2 must have two independent functional contact surfaces, one for binding to the effector enzyme and the other for dimerization of the Ca2+-free GCAP-2. We speculate that the ability to bind Ca2+ within the first EF-hand of GCAP-2 was lost in exchange for the ability to interact with the target cyclase.RESULTS AND DISCUSSIONBased on its NMR structure determined by Ames et al. (29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), GCAP-2 is similar to recoverin and neurocalcin and consists of two globular pairs of EF-hand structures connected by a "hinge" region between EF-2 and EF-3 (Fig. 1). Three EF-hands (EF-2, -3, and -4) contribute to the functional switch that causes GCAP-2 to undergo an "activator-to-inhibitor" transition when it binds Ca2+ (EC50Ca ∼ 200–300 nm, Hill coefficient ∼ 1.7–2.1, Ref. 8Dizhoor A.M. Hurley J.B. Methods. 1999; 19: 521-531Crossref PubMed Scopus (65) Google Scholar). Three Ca2+ ions bind to GCAP-2 with an apparentK d(Ca) of ∼300 nm and the cooperativity factor of 2 (29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Unlike these three EF-hands, the first EF-hand-related motif is short of several key side chain residues necessary for high affinity binding of Ca2+. To efficiently coordinate Ca2+ ion in the EF-hand loop by five side chain residues and one carbonyl oxygen of the main chain (36Strynadka N.C. James M.N. Annu. Rev. Biochem. 1989; 58: 951-998Crossref PubMed Google Scholar, 37Celio M.R. Pauls T.L. Shwaller B. Guidebook to the Calcium-binding Proteins.in: Celio M.R. Pauls T.L. Shwaller B. Oxford University Press, New York1996: 15-20Google Scholar, 38Kawasaki H. Kretsinger R.H. Protein Profile. 1994; 1: 343-517PubMed Google Scholar), it is required that the first Ca2+-coordinating position (X) of the 12-amino acid consensus motif be occupied by Asp, and both the positions 3 (Y) and 9 (−X) include oxygen-containing side chain residues (36Strynadka N.C. James M.N. Annu. Rev. Biochem. 1989; 58: 951-998Crossref PubMed Google Scholar). The absence of the first invariant Asp (replaced by Glu33) of the consensus motif and replacement of the obligatory oxygen-containing side chain residues by Cys35 (Y) and Phe41(−X) prohibit Ca2+ from binding within the loop structure of the EF-1 domain (16Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar).Although the EF-1 domain is unable to bind Ca2+, its amino acid sequence remains highly homologous between recoverin-like proteins, except for several variable residues that are specific for each particular member of the family (23Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). Also, regardless of its deviation from the EF-hand Ca2+ binding consensus motif, EF-1 retains an overall shape of a helix-loop-helix domain similar to other EF-hands (29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar).A previous observation (33Olshevskaya E.V. Boikov S. Ermilov A. Krylov D. Hurley J.B. Dizhoor A.M. J. Biol. Chem. 1999; 274: 10823-10832Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) indicated that the EF-1 was an essential part of the GCAP-2 molecule. Substitution of that domain in GCAP-2 with the corresponding segment from neurocalcin resulted in a loss of GCAP-2 activity. Yet the functional role of the EF-1 remained unclear. The uncertainty has been 2-fold. First, the retGC activator form of GCAP-2 can associate as a homodimer and dissociate in the presence of Ca2+. The inability of several GCAP-2 chimera mutants to form Ca2+-free dimers correlated with the lack of their ability to promote retGC activation in the absence of Ca2+ (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, the EF-1 could be involved in either target binding or dimerization of GCAP-2 (or both). Second, it could not be completely excluded that substitutions of a relatively large segment in the molecule, even with the corresponding fragment from a homologous protein, causes general misfolding of the protein. To further elucidate the possible functional role of the EF-1 domain in GCAP-2, we probed this region by single mutations that substituted some highly conserved (Cys35, Glu44) or variable (Lys30, Glu33, Phe41, His43, Phe48) amino acids (Fig. 1).We have found that activation of retGC at low Ca2+ concentrations is highly sensitive to even single substitutions in most of these amino acid residues (Fig.2, A–F). One of the invariant residues within the EF-1 motif of recoverin-like proteins is Cys35. Substitution of Cys35 with Ser (Fig.2 B) or Thr (data not shown) noticeably affects retGC activation, and replacing it with positively or negatively charged amino acids strongly suppresses the ability of GCAP-2 to activate the cyclase. The most dramatic effect has been observed in the case of negatively charged Asp that renders the mutant protein virtually inactive. Another fairly highly conserved amino acid residue, Glu44, is also essential for retGC activation because its substitution even with another acidic residue, Asp, causes a prominent decrease in activity of GCAP-2, whereas its substitution with a non-charged oxygen-containing side chain, Ser, completely inactivates the retGC stimulating activity of the Ca2+-free GCAP-2 (Fig. 2 E).Figure 2Activation of retinal membrane guanylyl cyclase by GCAP-2 and its mutants in various positions within the EF-1. A, Lys30 (K30G, ▴); B, Cys35 (C35S, ⋄; C35K, ♦; C35D, ▴); C, Phe41 (F41S, ▵; F41I, ■); D, His43 (H43Q, ×; H43E, ); E, Glu44 (E44S, ●; inset, E44D, ●);F, Phe48 (F48I, ■; F48V, ♦; F48S, ●);G, Tyr81 (Y81F, ♦; inset, Y81I, ♦); in A–G, wild type (WT) GCAP-2 marked (○); H, Glu33, samples contained 250 ng of bovine serum albumin (BSA) (a), recombinant GCAP-2 (b), or its mutants, E33Q (c) or E33K (d). retGC activity in reconstituted photoreceptor membranes was assayed as described under "Experimental Procedures" at free Ca2+ concentrations not higher than 6 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Similar to that, substitutions of several GCAP-2-specific amino acids had a profound negative effect on retGC stimulation by the Ca2+-free GCAP-2 (Fig. 2, A, C,D, and F). Some of the substitutions markedly or even completely suppressed cyclase stimulation (for example, K30G, F41I, H43E, H43Q, F48V, F48S).Apparently, some amino acid side chain residues within the EF-1 domain are not essential for the interaction with the cyclase (Fig. 2,G and H). For example, we did not find evidence that replacement of Glu33 with Gln, or even positively charged Lys, can seriously affect retGC activation. The side chain of Tyr81 (which is a part of the exiting helix in the second EF-hand) is located in close proximity to the amino acid residues of the EF-1 structure (Ref. 29Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). It can be replaced with the amino acids not found in this position in GCAPs, Ile or Phe, without a prominent effect on cyclase activation (Fig. 2 H).Why would different mutations within the EF-1 domain affect retGC activation? If the amino acid residues in the EF-1 were required for GCAP-2 binding to the cyclase, then one would expect to find mutations within this domain that reduce the apparent affinity of GCAP-2 in our retGC activation assay. Indeed, several substitutions that reduced GCAP-2 activity apparently decreased its affinity for the cyclase. For example, the C35T and the C35S GCAP-2 mutants had their EC50 values increased 2.5- and 6-fold, respectively, compared with the wild type GCAP-2, whereas at saturation (above 2.5 μm, data not shown) both GCAP-2 mutants fully activated retGC (to 103 and 100% of the wild type control level, data not shown). Similarly to that, the H43Q mutant had at least 10-fold higher EC50, but at saturation (above 10 μm GCAP-2) the cyclase stimulation reached 88% of the wild type control level.In several cases (i.e. C35D, C35K, F41I, E44S, F48S) retGC activation by the GCAP-2 mutants remained very low and did not reach saturation in the conditions of the assay. For that reason, we were unable to reliably evaluate the exact values of their EC50, but it appeared increased at least 5–10-fold. In addition to that, we found that some mutations affected the maximal level of retGC activation rather than EC50. The EC50 increase in the case of the F48V mutant was only within 2-fold, but the cyclase activation at saturation did not exceed 30% of the wild type control.The right shift of a dose dependence curve in case of the EF-1 mutants could be in a most simplified manner interpreted as a decrease in retGC binding affinity. It would be more difficult to explain the lower level of maximal cyclase activation found in some cases. At this point, we cannot offer any conclusive explanation for this phenomenon. Activation of the cyclase may require multiple steps, and various conformational changes in GCAP-2 may have to occur before retGC is activated. Perhaps the EF-1 domain may not only be involved in binding interactions with the cyclase but also through the intramolecular interactions influence other functional domains of GCAP-2 (for instance, EF-2) and thus affect the overall conformational switch in GCAP-2. However, our present results also strongly indicate that despite its inability to bind Ca2+, the N-terminal EF-hand domain in GCAP-2 is an essential part of the molecule that is important for targeting retGC.GCAP-2 can form a complex with guanylyl cyclase both in its Ca2+-free and Ca2+-loaded form (8Dizhoor A.M. Hurley J.B. Methods. 1999; 19: 521-531Crossref PubMed Scopus (65) Google Scholar, 39Laura R.P. Hurley J.B. Biochemistry. 1998; 37: 11264-11271Crossref PubMed Scopus (59) Google Scholar). Instead of activating the cyclase, Ca2+-loaded GCAP-2 inhibits basal activity of retGC in washed photoreceptor membranes (8Dizhoor A.M. Hurley J.B. Methods. 1999; 19: 521-531Crossref PubMed Scopus (65) Google Scholar), arguably by interfering with reversible dimerization of retGC (26Yu H. Olshevskaya E. Duda T. Seno K. Hayashi F. Sharma R.K. Dizhoor A.M. Yamazaki A. J. Biol. Chem. 1999; 274: 15547-15555Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, we tested the ability of some of the GCAP-2 mutants in the EF-1 domain to inhibit cyclase basal activity at saturating Ca2+ concentrations (Fig. 3). We have found that several mutants that have lower ability to stimulate retGC in their Ca2+-free form are also less efficient as inhibitors of the cyclase basal activity. This appears to be consistent with the EF-1 domain of GCAP-2 being involved in the interaction with the cyclase both in the absence and in the presence of Ca2+.Figure 3Inhibition of retGC basal activity by Ca2+-loaded GCAP-2 mutants at saturating free Ca2+ concentrations (∼10−5m). A, time course of cGMP synthesis in the presence of bovine serum albumin (BSA) (■), wtGCAP-2 (○), or C35D (▪). B, retGC activity as a function of Ca2+-loaded GCAP-2 (○) or its mutants: E44S (●), H43Q (×), H43E (♦), F41S (▴). The conditions of the assay are described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)It seems highly unlikely that different single amino acid substitutions within the EF-1 would all be a result of a general nonspecific misfolding of the protein. It is much more likely that these mutations directly affect the cyclase-binding site. However, both wild type and EF-1 mutants of GCAP-2 used in this study were isolated as Ca2+-loaded monomers (data not shown) using a previously described technique (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). At the same time, according to our previous observations, Ca2+-free GCAP-2 undergoes dimerization, which is likely to contribute to the cyclase regulation by promoting the interaction between two retGC subunits (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This Ca2+-sensitive dimerization of GCAP-2 apparently involves multiple regions in the GCAP-2 molecule (28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Hence, the GCAP-2 mutants tested in the present study could be inactive because of a nonspecific misfolding of the protein. On the other hand, if the EF-1 were specifically required for the Ca2+-sensitive dimerization of GCAP-2, rather than retGC binding, that could also account for the absence of their activity. However, in both cases we would expect that the inactive GCAP-2 mutants failed to form dimers in the absence of Ca2+. Contrary to that, all tested EF-1 mutants, even those that completely lost their ability to regulate the cyclase, were able to dimerize at low Ca2+ concentration (Fig.4, results with the rest of the GCAP-2 mutants are not shown, but all of them formed dimers in a Ca2+-free solution). These results strongly argue that 1) individual point mutations within the EF-1 domain do not cause general nonspecific misfolding of the protein and 2) these mutations specifically affect GCAP-2 interactions with retGC rather than the GCAP/GCAP interactions (as illustrated in Fig.5).Figure 4Ca2+-free GCAP-2 mutants within the EF-1 region remain capable of forming dimers. Purified recombinant proteins were analyzed using high resolution gel chromatography as described under "Experimental Procedures."a and b, wild type GCAP-2; c, F41I;d, E44S; e, F48S; f, C35D. Sample and elution buffer contained either 300 μm CaCl2(a) or 500 μm EGTA (b–f).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Putative role of EF-1 domain in GCAP-2 in retGC regulation. A, GCAP-2 binds to the cyclase both at high and low free Ca2+ concentrations. The release of Ca2+ provides a conformational switch that transforms GCAP-2 into its activator form and also stimulates GCAP/GCAP interactions. The activator GCAP-2 dimer enhances association between retGC subunits required for its catalytic activity (dimer-adapter hypothesis, Ref. 28Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). B, mutations within the EF-1 domain of GCAP-1 inactivate retGC regulation but do not abolish GCAP/GCAP dimer formation; therefore, it is most likely that the specific role of EF-1 is to make a contact surface for binding for the cyclase (or at least a part of such surface) in GCAP-2. Once the GCAP/retGC interactions are affected
Referência(s)