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The two types of photoreceptors in the vertebrate retina, rods and cones, mediate vision at dim and bright light, respectively. Cones produce small rapidly-decaying responses and are much less sensitive to light than rods. The molecular basis for the differences in physiology of rods and cones are poorly understood. The phototransduction cascades in rods and cones utilize homologous components including visual pigments, heterotrimeric G proteins (transducins), and cGMP-phosphodiesterases (PDE6) (1–3). In rods, a robust amplification in the signaling cascade is achieved due to a high rate of transducin activation by photolyzed rhodopsin (Meta II or R*) and rapid hydrolysis of cGMP by transducin-activated PDE6. Consistent with the low sensitivity of cones, the signal amplification in mouse cone phototransduction was found to be lower (4). The molecular differences between major cone and rod signaling proteins have been examined as a probable origin of the physiological differences (5–9). Current evidence does not implicate the signaling properties of rod and cone visual pigments. Expression of rhodopsin and red cone pigment in Xenopus cones and rods, respectively, yielded responses identical to those of native Xenopus photoreceptors (5). Recent physiological analysis points to an important role of transducin α-subunits in shaping specific photoresponses (7). Rods of GNAT2C transgenic mice with substitution of rod Gαt1 for cone Gαt2 displayed decreased sensitivity, reduced rate of activation, and accelerated recovery characteristic of cone photoreceptors (7). Reduced rate of cascade activation may result from a slower rate of R*-dependent activation of the Gαt2 complex with rod-specific Gβ1γ1 or less efficient activation of PDE6 by Gαt2 (10). Accelerated recovery of GNAT2C rods suggests faster GTP hydrolysis in the transition complex of Gαt2 with PDE6 and the RGS9-1 GTPase accelerating protein (GAP) complex (11). Thus, Gαt2 may have a higher intrinsic rate of GTP hydrolysis and/or a greater GTPase potentiation by the GAP complex than Gαt1. In contrast to well-characterized rod Gαt1, biochemical properties of Gαt2 have not been investigated. Native Gαt1 is readily available for biochemical analyses, whereas the sparsity of cones in mammalian retina impedes isolation of native Gαt2. In addition, a wealth of information about Gαt1 properties, including its interaction with R*, PDE6 and RGS9, was developed using robust bacterial expression of transducin-like Gαt1/Gαi1 chimeras (12–14). Here, we applied a chimera approach to analyze key signaling properties of Gαt2. A Gαt2/Gαi1 chimera (Gαt2′) was produced and investigated in comparison to analogous Gαt1/Gαi1 chimera Chi8 (13), termed hereafter Gαt1′. Gαt2′ and Gαt1′ are ~94% identical to Gαt2 and Gαt1, respectively, which is significantly greater than the degree of homology between Gαt2 and Gαt1 (~82%). Gαt2′ showed a large ~10-fold increase in spontaneous nucleotide exchange rate in comparison to Gαt1′. A series of chimeric and mutant Gαt subunits delineated Gαt2′ residues responsible for high intrinsic nucleotide exchange rate. In contrast, the rates of R*-dependent nucleotide exchange were comparable for Gαt2′ and Gαt1′ reconstituted with Gβ1γ1. Furthermore, Gαt2′ and Gαt1′ exhibited similar rates of intrinsic GTPase activity as well as similar acceleration of GTP hydrolysis by RGS9.

The interaction of phosphodiesterase 6 (PDE6) with its inhibitory Pgamma-subunits (Pgamma) is unparalleled among PDE families and is central to vertebrate phototransduction. The C-terminus of Pgamma occludes the active site of PDE6, thereby preventing hydrolysis of cGMP. In this study, we examine the determinants of this critical interaction using structure-based loss-of-function mutagenesis of a chimeric PDE5/PDE6 catalytic domain and gain-of-function mutagenesis of the PDE5 catalytic domain. This analysis revealed the key role of PDE6-specific residues within the catalytic domain M-loop-alpha-helix 15 region and suggested an important contribution of the H-loop-M-loop interface to the PDE6 inhibition by the Pgamma C-terminus. Identification of the determinants for the PDE6-Pgamma interaction offers insights into the evolution of the visual effector enzyme.

Photoreceptor rod and cone phosphodiesterases comprise the sixth family of cyclic nucleotide phosphodiesterases (PDE6). PDE6s have uniquely evolved as effector enzymes in the vertebrate phototransduction cascade. To understand the evolution of the PDE6 family, we have examined PDE6 in lamprey, an ancient vertebrate group. A single PDE6 catalytic subunit transcript was found in the sea lamprey Petromyzon marinus cDNA library. The lamprey PDE6 sequence showed a high degree of homology with mammalian PDE6 and equally distant relationships with the rod and cone enzymes. In contrast, two different PDE6 inhibitory Pgamma subunits, a cone-type Pgamma1 and a mixed cone/rod-type Pgamma2, have been identified in the lamprey retina. Immunofluorescence analysis demonstrated that Pgamma1 and Pgamma2 are expressed in the long and short photoreceptors of sea lamprey, respectively. The catalytic PDE6 subunit was present in the photoreceptors of both types and colocalized with the Pgamma subunits. Recombinant Pgamma1 and Pgamma2 potently inhibited trypsin-activated lamprey and bovine PDE6 enzymes. Our results point to a high degree of conservation of PDE6 genes during the vertebrate evolution. The apparent duplication of the Pgamma gene in the stem of vertebrate lineage may have been an essential component of the evolution of scotopic vision in early vertebrates.

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the vertebrate visual transduction cascade. The activity of rod PDE6 catalytic alpha- and beta-subunits is blocked in the dark by two inhibitory Pgamma-subunits. The inhibition is released upon light-stimulation of photoreceptor cells. Mutation H258N in PDE6beta has been linked to congenital stationary night blindness (CSNB) in a large Danish family (Rambusch pedigree) (Gal, A., Orth, U., Baehr, W., Schwinger, E., and Rosenberg, T. (1994) Nat. Genet. 7, 64-67.) We have analyzed the consequences of this mutation for PDE6 function using a Pgamma-sensitive PDE6alpha'/PDE5 chimera, Chi16. Biochemical analysis of the H257N mutant, an equivalent of PDE6betaH258N, demonstrates that this substitution does not alter the ability of chimeric PDE to dimerize or the enzyme's catalytic properties. The sensitivity of H257N to a competitive inhibitor zaprinast was also unaffected. However, the mutant displayed a significant impairment in the inhibitory interaction with Pgamma, which was apparent from a approximately 20-fold increase in the K(i) value (46 nM) and incomplete maximal inhibition. The inhibitory defect of H257N is not due to perturbation of noncatalytic cGMP binding to the PDE6alpha' GAF domains. The noncatalytic cGMP-binding characteristics of the H257N mutant were similar to those of the parent PDE6alpha'/PDE5 chimera. Since rod PDE6 in the Rambusch CSNB is a catalytic heterodimer of the wild-type PDE6alpha and mutant PDE6beta, Chi16 and H257N were coexpressed, and a heterodimeric PDE, Chi16/H257N, was isolated. It displayed two Pgamma inhibitory sites with the K(i) values of 5 and 57 nM. Our results support the hypothesis that mutation H258N in PDE6beta causes CSNB through incomplete inhibition of PDE6 activity by Pgamma, which leads to desensitization of rod photoreceptors.

Proteins containing G-protein regulatory (GPR) motifs represent a novel family of guanine nucleotide dissociation inhibitors (GDIs) for G(alpha) subunits from the Gi family. They selectively interact with the GDP-bound conformation of Gi(alpha) and transducin-alpha (Gt(alpha)), but not with Gs(alpha). A series of chimeric proteins between Gi(alpha)(1) and Gs(alpha) has been constructed to investigate GPR-contact sites on G(alpha) subunits and the mechanism of GPR-protein GDI activity. Analysis of the interaction of two GPR-proteins-AGS3GPR and Pcp2-with the chimeric G(alpha) subunits demonstrated that the GPR-Gi(alpha)(1) interface involves the Gi(alpha)(1) switch regions and Gi(alpha)(1)-144-151, a site within the helical domain. Residues within Gi(alpha)(1)-144-151 form conformation-sensitive contacts with switch III, and may directly interact with a GPR-protein or form a GPR-binding surface jointly with switch III. The helical domain site is critical to the ability of GPR-proteins to act as GDIs. Our data suggest that a mechanism of the GDI activity of GPR-proteins is different from that of GDIs for monomeric GTPases and from the GDI-like activity of G(betagamma) subunits. The GPR-proteins are likely to block a GDP-escape route on G(alpha) subunits.

In response to light, a photoreceptor G protein, transducin, activates cGMP-phosphodiesterase (PDE6) by displacing the inhibitory gamma-subunits (Pgamma) from the enzyme's catalytic sites. Evidence suggests that the activation of PDE6 involves a conformational change of the key inhibitory C-terminal domain of Pgamma. In this study, the C-terminal region of Pgamma, Pgamma-73-85, has been targeted for Ala-scanning mutagenesis to identify the point-to-point interactions between Pgamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. Pgamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzyme containing the Pgamma C-terminus-binding site of cone PDE. This analysis has revealed that in addition to previously characterized Ile86 and Ile87, important inhibitory contact residues of Pgamma include Asn74, His75, and Leu78. The patterns of mutant PDE5-conePDE6 enzyme inhibition suggest the interaction between the PgammaAsn74/His75 sequence and Met758 of the cone PDE6alpha' catalytic subunit. This interaction, and the interaction between the PgammaIle86/Ile87 and PDE6alpha'Phe777/Phe781 residues, is most consistent with an alpha-helical structure of the Pgamma C-terminus. The analysis of activation of PDE6 enzymes containing Pgamma mutants with Ala-substituted transducin-contact residues demonstrated the critical role of PgammaLeu76. Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with PgammaLeu76. This interaction introduces a bend into the alpha-helical structure of the Pgamma C-terminus, allowing transducin-alpha to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.

A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP >> Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.

The N-terminal regions of the heterotrimeric G-protein alpha-subunits represent one of the major Gbetagamma contact sites and have been implicated in an interaction with G-protein-coupled receptors. To probe the role of the N-terminal domain of transducin-alpha in G-protein function, a chimeric Gtialpha subunit with the 31 N-terminal Gtalpha residues replaced by the corresponding 42 residues of Gsalpha (Ns-Gtialpha) has been examined for the interaction with light-activated rhodopsin (R). Gtialpha displayed a somewhat higher R-stimulated rate of GTPgammaS binding relative to Ns-Gtialpha, suggesting modest involvement of the Gtalpha N-terminal sequence in recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtialpha was significantly faster (k(app) = 0.014 min(-)(1)) than that in Gtialpha (k(app) = 0.0013 min(-1)) as judged by the GTPgammaS binding rates. Substitution of 42 N-terminal residues of Gsalpha by the Gtalpha residues in a reciprocal chimera, Nt-Gsalpha, had an opposite effect-notable reduction in the intrinsic GTPgammaS-binding rate (k(app) = 0.0075 min(-)(1)) in comparison with Gsalpha (k(app) = 0.028 min(-)(1)). Residue Val30 (His41 in Gsalpha) within the N-terminal region of Gtalpha interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changes in the intrinsic nucleotide exchange rate in chimeric Galpha subunits might be attributed to this interaction, GtialphaVal30His, GtialphaIle339Ala, and Ns-GtialphaHis41Val mutants have been made and analyzed for basal GTPgammaS binding. GtialphaVal30His and GtialphaIle339Ala had increased GTPgammaS binding rates (k(app) = 0. 010 and 0.009 min(-)(1), respectively), whereas Ns-GtialphaHis41Val had a decreased GTPgammaS binding rate (k(app) = 0.0011 min(-)(1)) relative to their parent proteins. These results suggest that the coupling between the N-terminal and C-terminal domains of Gtalpha is important for maintaining a low nucleotide exchange rate in unstimulated transducin.

RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gs alpha. The Gs alpha residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gs alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction between Gs alpha and RGS proteins by substituting Gs alpha Asp229 with Ser and replacing the potential Gs alpha Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gs alpha. A single mutation of Gs alpha, Asp229Ser, rendered the Gs alpha subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq alpha-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gs alpha Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gs alpha Asp229Ser by approximately 5-fold with an EC50 value of 7.5 microM. Our findings demonstrate that Asp229 of Gs alpha represents a major barrier for Gs alpha interaction with known RGS proteins.

In photoreceptor cells of vertebrates, the GTP-bound alpha-subunit of rod G-protein, transducin (G(t alpha)), interacts with the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) to activate the effector enzyme. The GDP-bound G(t alpha) can also bind the Pgamma subunit, albeit with a lower affinity than G(t alpha)GTP. In this work, interactions between G(t alpha)GDP and Pgamma or Pgamma-24-45Cys labeled with the fluorescent probe 3-(bromoacetyl)-7-(diethylamino)coumarin (PgammaBC, Pgamma-24-45BC) have been investigated. Addition of G(t alpha)GDP to PgammaBC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence of Pgamma-24-45BC was enhanced by 2.3-fold. The Kd's for the G(t alpha)GDP binding to PgammaBC and Pgamma-24-45BC were 75 +/- 8 nM and 400 +/- 110 nM, respectively. The G(t betagamma) subunits had no notable effect on the binding of G(t alpha)GDP to PgammaBC or Pgamma-24-45BC, suggesting that Pgamma and G(t betagamma) bind to G(t alpha)GDP noncompetitively. The G(t alpha betagamma) interaction with the fluorescently labeled Pgamma was effectively blocked in the light-activated rhodopsin (R*)-G(t alpha betagamma) complex. Furthermore, addition of excess Pgamma or Pgamma-24-45 prevented binding of G(t alpha betagamma) to R*, indicating that the R* and Pgamma binding surfaces on G(t alpha betagamma) may overlap. It is likely that R* has a binding site within the alpha3-beta5 region of G(t alpha), which is a proposed site of G(t alpha)GDP binding to Pgamma-24-45. Alternatively, R* may induce conformational changes of the G(t alpha) alpha3-beta5 region such that the resulting structural changes alter the adjacent consensus sequence for the guanine ring binding of GDP/GTP(NKXD), and lead to a reduction in the affinity of G-protein for guanine nucleotides.

Mutations counterpart to dominant negative RasSer17Asn in the alpha-subunits of heterotrimeric G-proteins are known to also produce dominant negative effects. The mechanism of these mutations remains poorly understood. Here, we examined the effects and mechanism of the Ser43Cys and Ser43Asn mutants of transducin-like chimeric Gtalpha* in the visual signaling system. Our analysis showed that both mutants have reduced affinity for GDP and are likely to exist in an empty-or partially occupied-pocket state. S43C and S43N retained the ability to interact with Gtbetagamma and, as heterotrimeric proteins, bind to photoexcited rhodopsin (R*). The interaction with R* is unproductive as the mutants failed to bind GTPgammaS and become activated. S43C and S43N inhibited R*-dependent activation of Gtalpha* and Gtalpha, apparently by blocking R*. Finally, both Gtalpha* mutants lacked interaction with the gamma-subunit of PDE6, an effector protein in phototransduction. These results indicate that the S43C and S43N mutants of Gtalpha* are dominant negative inhibitors that bind and block the activated receptor in a mechanism that parallels that of RasSer17Asn. Dominant negative mutants of Gtalpha sequestering R*, such as S43C and S43N, may become useful instruments in probing the mechanisms of visual dysfunctions caused by abnormal phototransduction signaling.

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin alpha-subunit (Gtalpha) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the gamma-subunit of the effector enzyme, cGMP-phosphodiesterase (Pgamma). In contrast, Pgamma attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the Pgamma subunit exerts its effects on the GTPase activity of the Gtalpha-RGS complex via the C-terminal domain, Pgamma-63-87. The structural determinants that control the direction of Pgamma effects on the RGS-Gtalpha system are localized within the RGS domains. The addition of Pgamma caused an increase in the maximal stimulation of Gtalpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gtalpha GTPase activity by chimeric RGS16 and RGS9 proteins and Pgamma has been investigated. This analysis suggests that in addition to the differences in primary structures, the overall conformations of the RGS fold in RGS9 and RGS16 are likely to be responsible for the opposite effects of Pgamma on the RGS9 and RGS16 GAP activity. The RGS9 alpha3-alpha5 region constituted the minimal insertion of the RGS9 domain into RGS16 that reversed the inhibitory effect of Pgamma. A model of the RGS9 complex with Gtalpha shows the alpha3-alpha5 helices in RGS9 facing the proximate Pgamma binding site on Gtalpha. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by Pgamma involves a more rigid stabilization of the Gtalpha switch regions when Gtalpha is bound to both RGS9 and Pgamma.