Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the gamma-subunit.

The inhibitory interaction of phosphodiesterase-6 (PDE6) with its gamma-subunit (Pgamma) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pgamma-inhibitory peptide Pgamma(70-87) have been determined at 2.9 and 3.0 A, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pgamma and suggest the conformational change of Pgamma on transducin activation. Two variable H- and M-loops of PDE5/6cd form a distinct interface that contributes to the Pgamma-binding site. This allows the Pgamma C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pgamma-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pgamma(70-87)-binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

Heterologous expression of bovine rhodopsin in Drosophila photoreceptor cells.

PURPOSE: Vertebrate and invertebrate visual pigments are similar in amino acid sequence, structural organization, spectral properties, and mechanism of action, but possess different chromophores and trigger phototransduction through distinct biochemical pathways. The bovine opsin gene (Rho) was expressed in Drosophila, to examine the properties of a vertebrate opsin within invertebrate photoreceptor cells. METHODS: Transgenic Drosophila expressing the bovine opsin gene (Rho) in photoreceptors were created. Protein expression and cellular location of bovine rhodopsin was assessed by protein blots and immunofluorescence. The glycosylation state was determined by mobility profiles in SDS-PAGE before and after treatment with endoglycosidase. The rhodopsin chromophore was determined by HPLC-mass spectroscopy (MS) and the spectral properties by spectroscopy. The ability of the bovine rhodopsin to couple to Drosophila phototransduction components was assessed by electroretinography and to couple to vertebrate transducin by light-mediated GTPgammaS-binding assays. RESULTS: Rho showed stable expression even in the absence of endogenous Rh1 opsin and chromophore. It was correctly targeted to the rhabdomeric membranes. Rho remained glycosylated during the maturation process and possessed a distinct glycosylation pattern from that of native Rho. The Drosophila-expressed Rho associated with the 3-hydroxyretinal chromophore but failed to evoke an electroretinogram response from fly photoreceptors. However, the Drosophila-expressed Rho activated transducin in a light-dependent manner. CONCLUSIONS: Drosophila photoreceptors express a vertebrate rhodopsin as a functional visual pigment, but the expression does not activate the Drosophila phototransduction pathway. The system allows the characterization and comparison of vertebrate and invertebrate visual pigment properties in a common cell type.

Mutation R238E in transducin-alpha yields a GTPase and effector-deficient, but not dominant-negative, G-protein alpha-subunit.

PURPOSE: Certain forms of inherited and light-induced retinal degenerations are believed to involve excessive phototransduction signaling. A dominant-negative mutant of the visual G-protein, transducin, would represent a major tool in designing potential therapeutical strategies for this group of visual diseases. We thought to further investigate a novel mutant of the transducin-alpha subunit, R238E, that was recently reported to be a dominant-negative inhibitor of the rhodopsin/transducin/PDE visual system. METHODS: The R238E substitution was introduced into a tranducin-like chimeric Gtalpha*-subunit. The nucleotide-bound state of the Gtalpha*R238E mutant was assessed using the trypsin-protection assay. The ability of the Gtalpha*R238E mutant to interact with Gtbetagamma, couple to photoexcited rhodopsin (R*), and undergo R*-stimulated guanine nucleotide exchange was examined by a GTPgammaS binding assay. The GTPase activity of the mutant Gtalpha* and its interaction with RGS proteins was characterized in the steady-state and single turnover measurements of GTP hydrolysis. A binding assay utilizing the fluorescently-labeled gamma-subunit of PDE6 (Pgamma) was employed to monitor the effector function of Gtalpha*R238E. RESULTS: The Gtalpha*R238E mutant bound GDP and was capable of the AlF4--induced activational conformational change. The capacity of Gtalpha*R238E to couple to R* in the presence of Gtbetagamma was similar to that of Gtalpha*. However, the mutant GTPase activity was markedly impaired. This defect was further exacerbated by the diminished interactions of Gtalpha*R238E with the GAP proteins, RGS9 and RGS16. Another consequence of the mutation was the reduction in Gtalpha*R238E's affinity for Pgamma. CONCLUSIONS: Transducin mutant Gtalpha*R238E exists in a nucleotide-bound state and is fully capable of activational coupling to R*. This mutation results in a significant impairment of Gtalpha*'s ability to hydrolyze GTP and interact with the inhibitory subunit of PDE6. This phenotype is entirely inconsistent with that of a dominant-negative inhibitor as recently reported.

Probing rhodopsin-transducin interaction using Drosophila Rh1-bovine rhodopsin chimeras.

Invertebrate and vertebrate rhodopsins share a low degree of homology and are coupled to G-proteins from different families. Here we explore the utility of fly-expressed chimeras between Drosophila rhodopsin Rh1 and bovine rhodopsin (Rho) to probe the interactions between the invertebrate and vertebrate visual pigments and their cognate G-proteins. Chimeric Rh1 pigments carrying individual substitutions of the cytoplasmic loops C2 and C3 and the C-terminus with the corresponding regions of Rho retained the ability to stimulate phototranduction in Drosophila, but failed to activate transducin. Surprisingly, chimeric Rho containing the Rh1 C-terminus was fully capable of transducin activation, indicating that the C-terminal domain of vertebrate rhodopsins is not essential for the functional coupling to transducin.

Hsp40 couples with the CSPalpha chaperone complex upon induction of the heat shock response.

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

Mechanisms of dominant negative G-protein alpha subunits.

G-protein-coupled receptors (GPCRs) represent the largest class of membrane proteins and are the targets of 25-50% of drugs currently on the market. Dominant negative mutant Galpha subunits of heterotrimeric G-proteins have been extensively utilized to delineate G-protein signaling pathways and represent a promising new tool to study GPCR-dependent signaling in the CNS. There are different regions in various types of Galpha subunits in which mutations can give rise to a dominant negative phenotype. Such a mutant Galpha would compete with wild-type Galpha for binding to other proteins involved in the G-protein cycle and either block or reduce the response caused by wild-type Galpha. To date, there are three different mechanisms described for dominant negative Galpha subunits: sequestration of the Gbetagamma subunits, sequestration of the activated GPCR by the heterotrimeric complex, and sequestration of the activated GPCR by nucleotide-free Galpha. This review focuses on the development of dominant negative Galpha subunits, the different mechanisms used by various mutant Galpha subunits, and potential structural changes underlying the dominant negative effects.

Dominant negative mutants of transducin-alpha that block activated receptor.

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.

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 CSP(1-198) (full-length), CSP(1-112), and CSP(1-82) on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G alpha(s) and increases steady-state GTP hydrolysis. CSP(1-198) modulation of G alpha(s) was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP(1-112) was Hsc70-SGT-independent. CSP(1-112) preferentially associated with the inactive GDP-bound conformation of G alpha(s). Consistent with the stimulation of GTP hydrolysis, CSP(1-112) increased guanine nucleotide exchange of G alpha(s). The interaction of native G alpha(s) and CSP was confirmed by coimmunoprecipitation and showed that G alpha(s) associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the beta2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G alpha(s) 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.