Michael Natochin

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory γ-subunit of PDE (Pγ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin α-subunit, Gtα GDPAlF 4 − , while its binding to GtαGTPγS or GtαGDP was weak. hRGSr and Pγ did not compete for the interaction with Gtα GDPAlF 4 − . Affinity of the Pγ-Gtα GDPAlF 4 −interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of Gtα GDPAlF 4 −binding to Pγ labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PγBC). Binding of hRGSr to Gtα GDPAlF 4 −complexed with PγBC resulted in a maximal ∼40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for Gtα GDPAlF 4 −(K d 35 nm). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s−1. Addition of Pγ to the reconstituted system reduced the GTPase level accelerated by hRGSr (k cat 0.085 s−1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 μm rhodopsin these rates exceeded 0.7 s−1. Our data suggest that effects of hRGSr on transducin’s GTPase activity are attenuated by Pγ but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

Characterization of the G alpha(s) regulator cysteine string protein.

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Characterization of the Gαs Regulator Cysteine String Protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Rhodopsin Determinants for Transducin Activation A GAIN-OF-FUNCTION APPROACH

Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein transducin. Non-helical portions of the C2- and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a “gain-of-function” mutagenesis to identify rhodopsin residues critical for transducin activation. Mutant opsins with residues 140–148 (C2-loop), 229–244 (C3-loop), or 310–320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with poly-Ala substitutions in the C2- and C3-loops formed the 500-nm absorbing pigments but failed to activate transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Significant (∼50%) restoration of the rhodopsin/transducin coupling was achieved with re-introduction of residues Cys140/Lys141 and Arg147/Phe148 into the C2 template. The reverse substitutions of the C3-loop residues Thr229/Val230 and Ser240/Thr242/Thr243/Gln244 produced a pigment with a full capacity for transducin activation. The C4 template mutant was unable to bind 11-cis-retinal, and the presence of Asn310/Lys311 was required for correct folding of the protein. Subsequent mutagenesis of the C4-loop revealed the role of Phe313 and Met317. On the background of Asn310/Lys311, the inclusion of Phe313 and Met317 produced a mutant pigment with the potency of transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for transducin activation.

Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist‐bound G protein‐coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein α‐subunit (Gα) followed by the release of GαGTP and Gβγ which, subsequently, activate their targets. The C‐terminal regions of Gα subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR‐induced conformational change is communicated from the Gα C‐terminus, via the α5 helix, to the nucleotide‐binding β6/α5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C‐terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three‐turn α‐helical segment was inserted between the 11 C‐terminal residues and the α5 helix of Gαt‐like chimeric Gα, Gαti. The mutant Gα subunits with the Gly‐loop (GαtiL) and the extended α5 helix (GαtiH) retained intact interactions with Gβγt, and displayed modestly reduced binding to R*. GαtiH was capable of efficient activation by R*. In contrast, R* failed to activate GαtiL, suggesting that the Gly‐loop absorbs a conformational change at the C‐terminus and blocks G protein activation. Our results provide evidence for the role of Gα C‐terminus/α5 helix/β6/α5 loop route as a dominant channel for transmission of the GPCR‐induced conformational change leading to G protein activation.

Rhodopsin Recognition by Mutant Gsα Containing C-terminal Residues of Transducin

The C-terminal regions of the heterotrimeric G protein α-subunits play key roles in selective activation of G proteins by their cognate receptors. In this study, mutant Gsα proteins with substitutions by C-terminal residues of transducin (Gtα) were analyzed for their interaction with light-activated rhodopsin (R*) to delineate the critical determinants of the Gtα/R* coupling. In contrast to Gsα, a chimeric Gsα/Gtα protein containing only 11 C-terminal residues from transducin was capable of binding to and being potently activated by R*. Our results suggest that Cys347 and Gly348 are absolutely essential, whereas Asp346 is more modestly involved in the Gt activation by R*. In addition, the analysis of the intrinsic nucleotide exchange in mutant Gsα indicated an interaction between the C terminus and the switch II region in Gtα·GDP. Mutant Gsα containing the Gtα C terminus and substitutions of Asn239and Asp240 (switch II) by the corresponding Gtα residues, Glu212 and Gly213, displayed significant reductions in spontaneous guanosine 5′-O-(3-thiotriphosphate)-binding rates to the levels approaching those in Gtα. Communication between the C terminus and switch II of Gtα does not appear essential for the activational coupling between Gt and R*, but may represent one of the mechanisms by which Gα subunits control intrinsic nucleotide exchange.

Roles of the Transducin α-Subunit α4-Helix/α4-β6 Loop in the Receptor and Effector Interactions

The visual GTP-binding protein, transducin, couples light-activated rhodopsin (R*) with the effector enzyme, cGMP phosphodiesterase in vertebrate photoreceptor cells. The region corresponding to the α4-helix and α4-β6 loop of the transducin α-subunit (Gtα) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the α4-β6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the α4-β6 loop in the R*-Gtα interface and suggests that the Gtα residues Arg310 and Asp311 are involved in the interaction with R*. These residues are likely to contribute to the specificity of the R* recognition. Contrary to the evidence previously obtained with synthetic peptides of Gtα, our data indicate that none of the α4-β6 residues directly or significantly participate in the interaction with and activation of phosphodiesterase. However, Ile299, Phe303, and Leu306 form a network of interactions with the α3-helix of Gtα, which is critical for the ability of Gtα to undergo an activational conformational change. Thereby, Ile299, Phe303, and Leu306play only an indirect role in the effector function of Gtα.

An interface of interaction between photoreceptor cGMP phosphodiesterase catalytic subunits and inhibitory gamma subunits.

Cyclic guanosine 5′-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE γ subunits (Pγs) block catalytic activity of PDE-α and -β subunits (Pαβ) in the dark. The primary regions of Pγ involved in the interaction with Pαβ are a central polycationic region, Pγ-24-45, and a C-terminal region of Pγ. Recently, we have shown that the C-terminal region of Pγ, which is the major Pγ inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE α subunit that binds to the central polycationic domain of Pγ. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Pα-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pγ. In addition, Pα-461-553 binds to the Pγ-24-45 region (Kd, 7 μM), as measured by a fluorescent increase in a Pγ-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Pα-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Pα (Pα-517-541) effectively suppressed inhibition of PDE activity by Pγ and bound to Pγ-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 μM). Pα-517-541 also competes with the activated rod G-protein α-subunit for binding to Pγ labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin α-guanosine 5′-triphosphate and Pα-517-541 for binding to the Pγ-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.

Identification of Effector Residues on Photoreceptor G Protein, Transducin

Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric Gαt/Gαi1 α-subunit (Gαt/i), we identified Gαt residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile208 in the switch II region directly interacts with the effector in the active GTP-bound conformation of Gαt. Residues Arg201, Arg204, and Trp207are essential for the conformation-dependent Gαt/effector interaction either via direct contacts with the inhibitory PDE γ-subunit or by forming an effector-competent conformation through the communication network between switch II and the switch III/α3-helix domain of Gαt. Residues His244 and Asn247 in the α3 helix of Gαt are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the Gαt/i chimera with the ability to bind PDE γ-subunit and stimulate PDE activity approaching that of native Gαt. Comparative analysis of the interactions of Gαt/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/Gα/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.

Interaction of transducin-α with LGN, a G-protein modulator expressed in photoreceptor cells

LGN and activator of G-protein signaling 3 (AGS3) belong to the class of G-protein modulators containing G-protein regulatory motifs (GPR proteins). Evidence for the functions of these molecules has only started to emerge. Immunostaining of mouse retina cross-sections and serial tangential sectioning of the retina combined with immunoblot analysis revealed that LGN is expressed in the inner segments of photoreceptor cells. Double immunolabeling demonstrated that, following light-dependent translocation from the outer segments, the α-subunit of the visual G-protein transducin (Gtα) colocalizes with LGN in the basal part of the inner segments. LGN and Gtα coprecipitate from the retinal extracts, supporting the notion of the interaction between the proteins. Furthermore, the GPR domain of LGN potently inhibits receptor-mediated guanine nucleotide exchange and steady-state GTPase activity of transducin. The localization and interaction with Gtα suggest LGN roles in modulation of transducin translocation and other photoreceptor cell functions.