Characterization of the Gαs Regulator Cysteine String Protein

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including G␣ q and G␣ s regulation of neurotransmission, G␣ idependent mitotic spindle positioning during (asymmetric) cell division, and G␣ olf -dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for G␣ i /␣ q /␣ 12/13 subunits. Ric-8B potentiated G s signaling presumably as a G␣ s -class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be G␣ subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed G␣ s and lesser amounts of G␣ q and G␣ 13 . Ric-8BFL stimulated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding to these subunits and G␣ olf , whereas the Ric-8B⌬9 isoform stimulated G␣ s short GTP␥S binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V max of G␣ s steady state GTP hydrolysis and the apparent K m values of GTP binding to G␣ s from ϳ385 nM to an estimated value of ϳ42 M. Directionality of the Ric-8BFL-catalyzed G␣ s exchange reaction was GTP-dependent. At sub-K m GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free G␣ s tightly, and near-K m GTP levels were required to dissociate the Ric-8B⅐G␣ nucleotide-free intermediate to release free Ric-8B and G␣-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce G␣-GTP in cells.Heterotrimeric G proteins transduce signals received from ligand-bound G protein-coupled receptors (GPCRs) 2 to intracellular effector enzymes. Agonist-bound GPCRs activate coupled G protein heterotrimers by accelerating the rate of GDP for GTP exchange on the G␣ subunit (1). The understanding of G protein signaling pathway complexity expanded beyond this traditional paradigm when modulatory proteins that regulate G protein activation apart from receptors were uncovered and characterized (2-7).One well characterized non-receptor G protein activator is Ric-8 (resistance to inhibitors of cholinesterase 8A). ric-8 was first identified in a genetic screen devised to find mutants of genes that positively regulated Caenorhabditis elegans neurotransmission (8). Through genetic epistasis analyses, it was predicted that ric-8 action was elicited upstream of G␣ q and/or G␣ s to regulate divergent G protein signaling outputs (8 -10). Ric-8 was first linked physically to G proteins when two homologous mammalian Ric-8 proteins (so-named Ric-8A and Ric-8B) were isolated in yeast two hybrid screens using G␣ o and G␣ s baits, respectively. Ric-8A interacted with G␣ i/o , G␣ q , and G␣ 13 subunits (7). Ric-8B interacted with G␣ s and G␣ q (7, 11). Evidence of a preferred interaction of Ric-8A with G␣ i -GDP led to experimentation showing that Ric-8A was a guanine nucleotide exchange factor for the monomeric G␣...

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confidence: 99%

Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

Chan

Gabay

Wright

et al. 2011

“…This same Csp-Hsc70-small glutamine-rich tetratricopeptide repeat-containing protein complex also interacts with heterotrimeric G-proteins. In this context Csp functions as a guanine-nucleotide exchange factor for G␣ S , which leads to stimulation of G-protein-dependent signaling and to G-protein-mediated inhibition of N-type Ca 2ϩ channels (20,21). Our laboratory previously established a novel role for Csp at endoplasmic reticulum (ER) membranes in modulating the trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR).…”

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confidence: 99%

Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Schmidt

Watts

Aridor

et al. 2009

Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/ Hsp70 and CHIP-mediated CFTR degradation.Cysteine string protein (Csp, DnaJC5) 2 is a member of the DnaJ/Hsp40 protein chaperone family (1, 2). Csp contains a short N-terminal sequence, followed by the J-domain, a linker region, and the central cysteine-rich region, which gives these proteins their name. The cysteine residues in this region are post-translationally modified by the palmitate groups that serve for the membrane attachment of Csp (3). This structure is followed by a C-terminal domain, which is the most variable region among the three Csp paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic junctions of Drosophila neurons (4). Csps are expressed at high levels in neurons and in cell types involved in regulated exocytosis (5-7), where they have been shown to govern exocytic secretory functions. For example, depolarizationinduced synaptic vesicle exocytosis is impaired in neurons from Csp-deficient Drosophila (8), and Csp overexpression produced decreases in stimulated insulin release from ␤-cells and stimulated catecholamine release from chromaffin cells (9 -11). Deletion of the Csp gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder characterized by developing neurodegenerative changes (12, 13).As for other Hsp40 homologs, the J-domain is responsible for the ability of Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity (14). Similar to other chaperone proteins, Csp can be found in many different protein complexes in the...

“…Recently, a few non-receptor GEFs have been described, i.e. AGS1 (6), Ric-8 (7,8), CSP␣ (9), and Arr4 (10). In contrast to GPCRs, these non-receptor GEFs are structurally unrelated, and their physiological roles are just beginning to be elucidated (8,(11)(12)(13).…”

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confidence: 99%

A Structural Determinant That Renders Gαi Sensitive to Activation by GIV/Girdin Is Required to Promote Cell Migration

Garcia‐Marcos

Ghosh

Ear

et al. 2010

210

Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G␣ i3 necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G␣ i subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G␣ i3 is a better substrate for GIV than the highly homologous G␣ o . We identified Trp-258 in the G␣ i subunit as a novel structural determinant for GIV binding by comparing GIV binding to G␣ i3 /G␣ o chimeras. Mutation of Trp-258 to the corresponding Phe in G␣ o decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G␣-binding partners, i.e. G␤␥, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/ RGS19 (a GTPase-activating protein), and LPAR1 (a G proteincoupled receptor). Activation of G␣ i3 by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G␣ i3 W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G␣ i3 by GIV is essential for biological functions associated with G␣ i3 activation. In conclusion, we have discovered a novel structural determinant on G␣ i that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G␣ i and that represents an attractive target site for designing small molecules to disrupt the G␣ i -GIV interface for therapeutic purposes.