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.

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.

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.

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)

Small G proteins of the Rho/Rac/Cdc42 family are associated with lipid membranes through their prenylated C termini. Alternatively, these proteins form soluble complexes with GDI proteins. To assess how this membrane partitioning influences the activation of Rac by guanine nucleotide exchange factors, GDP-to-GTP exchange reactions were performed in the presence of liposomes using different forms of Rac-GDP. We show that both non-prenylated Rac-GDP and the soluble complex between prenylated Rac-GDP and GDI are poorly activated by the Dbl homology-pleckstrin homology (DH-PH) domain of the exchange factor Tiam1, whereas prenylated Rac-GDP bound to liposomes is activated about 10 times more rapidly. Sedimentation experiments with liposomes reveal that the DH-PH region of Tiam1 forms, with nucleotide-free prenylated Rac, a membrane-bound complex from which GDI is excluded. Taken together, these experiments demonstrate that the dissociation of Rac-GDP from GDI and its translocation to membrane lipids favor DH-PH-catalyzed nucleotide exchange because the steric hindrance caused by GDI is relieved and because the membrane environment favors functional interaction between the DH-PH domain and the small G protein.Small G proteins of the Rho family, including Rho, Rac, and Cdc42p, undergo two interdependent cycles. First, they cycle between inactive (GDP) and active (GTP) conformations through the catalytic action of guanine exchange factors (GEF) 1 and GTPase-activating proteins. Second, they cycle between cytosolic and membrane-associated forms through the action of GDI proteins (1-3). How these two cycles are coupled is critical for the proper interaction of Rho proteins with their targets. It is generally assumed that Rho proteins must be in the GTP conformation and associated to membranes to trigger a cellular response.Dbl homology (DH) domains catalyze the exchange of guanine nucleotides on small G proteins of the Rho family (4, 5). The DH domain is an all-␣-helix fold that is systematically followed by a pleckstrin homology (PH) domain. The DH-PH tandem is thus the hallmark of Rho GEFs, which otherwise display variable domain composition and organization. Recent structural and mutagenesis studies on DH-PH domains, either isolated or in complex with Rho proteins, have given insights into the mechanism by which these domains promote the release of the bound nucleotide (6 -10). One of the best-studied examples is Tiam, a GEF for Rac. The DH domain of Tiam makes extensive contacts with the switch I and II regions of Rac and modifies the magnesium, sugar, and guanine basebinding regions to destabilize the bound nucleotide (9). In addition, structure-based mutagenesis studies show that the specificity between functional GEF/Rho pairs relies on a few residues at the DH/switch II interface (11-13). Binding to switch I and II regions is a general property of GEFs for small G proteins (14).Despite these spectacular progresses, one aspect of the activation of Rho proteins by DH domains remains obscure: how does the nucleotide ...

A BSTR ACTGuanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca 2؉ , 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 Ca 2؉ -sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.In photoreceptor cells, photoactivation of rhodopsin or cone visual pigment results in a transient decrease in the concentrations of Ca 2ϩ and cGMP. These receptors and second messengers are linked through a cascade of specific activation͞ inactivation reactions in phototransduction (1, 2). The levels of Ca 2ϩ and cGMP are strictly controlled and interconnected. cGMP is a gating ligand of the plasma membrane cation channels that are permeable to Ca 2ϩ ions. After cGMP is hydrolyzed, the efflux of Ca 2ϩ exceeds the influx, resulting in decreased [Ca 2ϩ ] within the cell. The lowering of [Ca 2ϩ ] triggers production of cGMP through activation of a photoreceptor-specific particulate guanylate cyclase (GC) (3). The Ca 2ϩ sensitivity of GC (e.g., the higher activity at low levels of [Ca 2ϩ ]) is mediated by one or more Ca 2ϩ -binding proteins, termed guanylate cyclase-activating proteins (GCAPs) (4, 5).Two photoreceptor-specific GCs, GC1 and GC2, have been cloned (6-9). Although the localization of GC1 to rod and cone outer segments and synaptic terminals was established by both biochemical and immunocytochemical methods (5,(10)(11)(12)(13)(14), the localization of GC2 within photoreceptors is not known. Two GCAPs that stimulate GC1 and GC2 have also been cloned (15-17). There is abundant evidence that GCAP1 activates photoreceptor GC: (i) GCAP1 was isolated from rod outer segments (ROS) (4, 18); (ii) GCAP1 mRNA was found in the myoid region of rod and cone photoreceptor cells (15, 19); (iii) GCAP1 was localized to rod and cone outer segments and to some cone somata and synaptic terminals by immunocytochemistry (17, 18); (iv) cross-linking techniques revealed that GCAP1 was complexed with GC1 (20). In addition, the finding that GC1 is not expressed in the retina of the rd...

Guanylyl cyclase-activating proteins (GCAPs

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 Ca 2ϩ -binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca 2ϩ ], but inhibits GC in high [Ca 2ϩ ], 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 Ca 2ϩ -binding motif of GCIP is disabled for Ca 2ϩ 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 Ca 2ϩ -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 [Ca 2ϩ ], competing with GCAP1 and GCAP2 for GC regulatory sites.