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).

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.

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.

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.

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.

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.

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.