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2.
J Biol Chem ; 274(28): 19829-37, 1999 Jul 09.
Article in English | MEDLINE | ID: mdl-10391927

ABSTRACT

Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding proteins belonging to the calmodulin superfamily. Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca2+-binding loops were replaced by reporter Trp residues. Using fluorescence spectroscopy and biochemical assays, we found that binding of Ca2+ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energy Ea = 9.3 kcal/mol. When Tyr99 adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys99 is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurements indicated that GCAP1 rapidly loses its bound Ca2+ (k-1 = 72 s-1 at 37 degrees C) and was estimated to associate with Ca2+ at a rate (k1 > 2 x 10(8) M-1 s-1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium/pharmacology , Protein Conformation , Tryptophan/genetics , Animals , Cattle , Guanylate Cyclase/metabolism , Guanylate Cyclase-Activating Proteins , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Rod Cell Outer Segment/enzymology , Spectrometry, Fluorescence , Tryptophan/chemistry
3.
Proc Natl Acad Sci U S A ; 95(25): 15014-9, 1998 Dec 08.
Article in English | MEDLINE | ID: mdl-9844007

ABSTRACT

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.


Subject(s)
Calcium/metabolism , Retina/physiology , Rhodopsin/physiology , Type C Phospholipases/pharmacology , Animals , Cattle , Phosphorylation , Type C Phospholipases/metabolism
4.
J Neurosci ; 18(9): 3195-205, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9547228

ABSTRACT

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events.


Subject(s)
Calcium/physiology , Cilia/enzymology , Cyclic AMP/physiology , Guanylate Cyclase/physiology , Olfactory Receptor Neurons/physiology , Animals , Calcium-Binding Proteins/physiology , Enzyme Activation , Guanylate Cyclase-Activating Proteins , Kinetics , Nerve Tissue Proteins/physiology , Odorants , Particle Size , Phosphorylation , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology
5.
Eur J Biochem ; 252(3): 591-9, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9546678

ABSTRACT

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+-binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+-binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Retina/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA Primers , Gene Library , Guanylate Cyclase-Activating Proteins , Mammals , Molecular Sequence Data , Photoreceptor Cells/metabolism , Protein Structure, Secondary , Rana pipiens , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Rod Cell Outer Segment/metabolism , Sequence Alignment , Sequence Homology, Amino Acid
6.
Biochemistry ; 36(14): 4295-302, 1997 Apr 08.
Article in English | MEDLINE | ID: mdl-9100025

ABSTRACT

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/metabolism , Guanylate Cyclase/metabolism , Retinal Cone Photoreceptor Cells/enzymology , Retinal Rod Photoreceptor Cells/enzymology , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Animals , Calcium/pharmacology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/pharmacology , Cattle , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/drug effects , Gene Expression , Guanylate Cyclase-Activating Proteins , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Retina/enzymology , Rod Cell Outer Segment/enzymology , Spectrometry, Fluorescence
7.
Proc Natl Acad Sci U S A ; 94(9): 4727-32, 1997 Apr 29.
Article in English | MEDLINE | ID: mdl-9114059

ABSTRACT

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.


Subject(s)
Calcium-Binding Proteins/isolation & purification , Nerve Tissue Proteins/isolation & purification , Photoreceptor Cells/chemistry , Retina/chemistry , Animals , Calcium-Binding Proteins/genetics , Cattle , Guanylate Cyclase-Activating Proteins , Humans , Immunohistochemistry , In Situ Hybridization , Macaca fascicularis , Nerve Tissue Proteins/genetics , Neurons/chemistry , Neurons/ultrastructure , Photoreceptor Cells/anatomy & histology , Retina/anatomy & histology , Retinal Cone Photoreceptor Cells/chemistry , Retinal Cone Photoreceptor Cells/ultrastructure , Retinal Rod Photoreceptor Cells/chemistry , Retinal Rod Photoreceptor Cells/ultrastructure , Species Specificity , Synapses/chemistry , Synapses/ultrastructure
8.
Biochemistry ; 33(51): 15215-22, 1994 Dec 27.
Article in English | MEDLINE | ID: mdl-7803383

ABSTRACT

We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.


Subject(s)
GTP Phosphohydrolases/metabolism , Rod Cell Outer Segment/enzymology , Transducin/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Animals , Cattle , Cyclic GMP/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Triphosphate/metabolism , In Vitro Techniques , Kinetics
9.
FEBS Lett ; 343(3): 183-7, 1994 May 02.
Article in English | MEDLINE | ID: mdl-8174698

ABSTRACT

The gamma-subunit of the cGMP-phosphodiesterase (PDE gamma) of retinal rods forms a tight complex with the activated alpha-subunit of transducin (Gt alpha GTP gamma S). We observe that while PDE gamma is not the physiological effector of other G alpha subtypes, it can still detectably interact with them. This interaction is strong with Gi1 alpha and Gi3 alpha (Kd approximately 10 nM) and weaker with Go alpha and Gs alpha (Kd approximately 1 microM). For all these G alpha subtypes, similar intrinsic fluorescence changes are observed upon PDE gamma binding. Moreover, similar relative decreases in affinity are obtained when the GDP forms of Gi1 alpha, Gi3 alpha or Gt alpha are used in lieu of the GTP forms. This points to a conserved GTP-dependent effector-interaction domain.


Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/metabolism , Signal Transduction , Transducin/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/chemistry , Amino Acid Sequence , Animals , Cattle , Molecular Sequence Data , Protein Conformation , Rats , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Transducin/chemistry
10.
EMBO J ; 12(11): 4191-8, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8223434

ABSTRACT

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.


Subject(s)
GTP-Binding Proteins/metabolism , Rod Cell Outer Segment/metabolism , Signal Transduction , Transducin/metabolism , Tryptophan/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Aluminum Compounds/metabolism , Amino Acid Sequence , Animals , Baculoviridae , DNA Mutational Analysis , Enzyme Activation , Fluorescence , Fluorides/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Moths/cytology , Protein Conformation , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Transducin/genetics , Tryptophan/genetics
11.
Biochemistry ; 32(33): 8636-45, 1993 Aug 24.
Article in English | MEDLINE | ID: mdl-8395212

ABSTRACT

In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Rod Cell Outer Segment/metabolism , Transducin/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , Amino Acid Sequence , Animals , Base Sequence , Binding, Competitive , Cattle , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Kinetics , Macromolecular Substances , Mathematics , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Recombinant Proteins/metabolism , Restriction Mapping , Retina/metabolism , Tryptophan
12.
Biochemistry ; 32(33): 8646-53, 1993 Aug 24.
Article in English | MEDLINE | ID: mdl-8395213

ABSTRACT

The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Guanosine Triphosphate/metabolism , Rod Cell Outer Segment/metabolism , Transducin/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Animals , Cattle , Cholera Toxin/metabolism , Filtration/instrumentation , Filtration/methods , GTP Phosphohydrolases/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Macromolecular Substances , Spectrometry, Fluorescence , Transducin/isolation & purification
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