The inhibitory interaction of phosphodiesterase-6 (PDE6) with its c-subunit (Pc) 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 Pc-inhibitory peptide Pc 70À87 have been determined at 2.9 and 3.0 Å , respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pc and suggest the conformational change of Pc on transducin activation. Two variable H-and M-loops of PDE5/6cd form a distinct interface that contributes to the Pc-binding site. This allows the Pc 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 Pc-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 Pc 70À87 -binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

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 Nterminal J domain characteristic of the DnaJ/Hsp40 cochaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca 2؉ 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␣ s and increases steady-state GTP hydrolysis. CSP 1-198 modulation of G␣ 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␣ s . Consistent with the stimulation of GTP hydrolysis, CSP 1-112 increased guanine nucleotide exchange of G␣ s. The interaction of native G␣ s and CSP was confirmed by coimmunoprecipitation and showed that G␣ s associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the ␤ 2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G␣ 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.G proteins constitute a family of heterotrimeric GTP-binding proteins that act as transducers in a variety of transmembrane signaling systems. G proteins are composed of ␣, ␤, and ␥ subunits that dissociate into G␣ and G␤␥ upon activation. Activation of G proteins involves an exchange of GDP for GTP on G␣ subunits and the release of GTP-bound G␣ and G␤␥ to interact with effector molecules. Effector molecules include adenylate cyclase, phospholipases, phosphodiesterases, and ion channels. Several modulators of G proteins have been identified and are thought to play crucial roles in the kinetics of G protein signaling (e.g. guanine nucleotide exchange factors (GEFs), 1 guanine nucleotide dissociation inhibitors, and GTPase-activating proteins). Our previous work has identified cysteine string protein (CSP) as a novel modulator of G proteins (1-3). Although the functional parallels between CSP and established G protein modulators are evident, the molecular mechanism by which CSP regulates G proteins is not yet known.CSPs are secretory vesicle proteins of 34 kDa that contain three conserved domains: a J domain, a linker domain, and a cysteine string region (Fig. 1A). The J domain is a 70-amino acid region of homology shared by DnaJ (a well characterized bacterial co-chaperone) and many otherwise unrelated eukaryotic proteins. The linker domain is a 30-amino acid ...

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.

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 (CSPα). CSPα 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 CSPα chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Gαs. 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 CSPα complex. Association of Hsp40 with CSPα decreases CSPα-CSPα dimerization and enhances the CSPα-induced increase in steady state GTP hydrolysis of Gαs. This newly identified CSPα-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 CSPα in neuroprotection.

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.