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Abstract: L7/Pcp-2 is a GoLoco domain protein encoded by a Purkinje cell dendritic mRNA. Although biochemical interactions of GoLoco proteins with Gαo and Gαi are well documented, little is known about effector function modulation resulting from these interactions. The P-type Ca2+ channels might be physiological effectors of L7 because (1) they are the major voltage-dependent Ca2+ channels (VDCC) that modulate Purkinje cell output and (2) they are regulated by Gi/o proteins. As a first step towards validating this hypothesis and to further understand the possible physiological effect of L7 protein and its two isoforms, we have coexpressed Cav2.1 channels and κ-opioid receptors (KORs) with varying amounts of L7A or L7B in Xenopus oocytes and measured ionic currents by two-electrode voltage clamping. Without receptor activation L7 did not alter the Ca2+ channel activity. With tonic and weak activation of the receptors, however, the Ca2+ channels were inhibited by 40–50%. This inhibition was enhanced by low, but dampened by high, expression levels of L7A and L7B and differences were observed between the two isoforms. The enhancing effect of L7 was occluded by overexpression of Gβγ, whereas the disinhibition was antagonized by overexpression of Gαo. We propose that L7 differentially affects the Gα and Gβγ arms of receptor-induced Gi/o signaling in a concentration-dependent manner, through which it increases the dynamic range of regulation of P/Q-type Ca2+ channels by Gi/o protein-coupled receptors. This provides a framework for designing further experiments to determine how dendritic local fluctuations in L7 protein levels might influence signal processing in Purkinje cells. [Copyright &y& Elsevier]

ALS2, the causative gene product for juvenile recessive amyotrophic lateral sclerosis (ALS2), is a guanine-nucleotide exchange factor for the small GTPase Rab5. Here, we report a novel ALS2 homologous gene, ALS2 C-terminal like (ALS2CL), which encodes a 108-kD ALS2CL protein. ALS2CL exhibited a specific but a relatively weak Rab5-GEF activity with accompanying rather strong Rab5-binding properties. In HeLa cells, co-expression of ALS2CL and Rab5A resulted in a unique tubulation phenotype of endosome compartments with significant colocalization of ALS2CL and Rab5A. These results suggest that ALS2CL is a novel factor modulating the Rab5-mediated endosome dynamics in the cells. [Copyright &y& Elsevier]

We have identified a new guanine-nucleotide exchange factor, P-Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P-Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)-trisphosphate and the βγ subunits of heterotrimeric G proteins in vitro and in vivo. P-Rex2 has structure, activity and regulatory properties similar to P-Rex1 but has divergent tissue distribution, as P-Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein-coupled receptors and phosphoinositide 3-kinase. [Copyright &y& Elsevier]

Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. When the objective is analysis of similarities and differences in the enzyme''s active site, there is an inherent problem in using the same domains for the superimposition. In this work we use a comparative approach termed here “Substrate Directed SuperImposition” (SDSI). It entails the superimposition of multiple protein–substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate''s point of view. Our analysis extends previous usage of similar approaches to comparison of enzyme catalytic machineries.We applied SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction that controls the signaling activity of this important family. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in this reaction, the reorientation of the critical arginine and glutamine. Additionally, we ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay the possible catalytic roles of the Lys16 residue in Ras GTPase. SDSI demonstrated that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12→Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Remarkably, SDSI also revealed similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinase. Taken together, our results promote the use of SDSI to compare the catalytic machineries of both similar and different classes of enzymes. [Copyright &y& Elsevier]

The monomeric GTPase Rac and the lipid kinase phosphoinositide 3-kinase (PI3K) are intracellular signalling enzymes that each regulate a huge range of cellular functions. Their signalling pathways overlap. Several pathways lead from PI3K activation via the production of the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) to the activation of guanine-nucleotide exchange factors (GEFs) that activate Rac. Vice versa, Rac can also stimulate the activation of PI3K, although the mechanism for this is unclear. We review here the evidence that links PI3K and Rac signalling pathways. [Copyright &y& Elsevier]

Sequestration of G protein-coupled receptors from the cell surface is a commonly observed phenomenon following agonist-stimulation. This process is now believed to be important for receptor resensitization as well as for signal transduction. Over the years, numerous studies have aimed at understanding the molecular mechanisms underlying internalization. Proteins such as the G protein-coupled receptor kinases (GRKs) and the ß-arrestins, which were initially characterized as desensitizing molecules, have been shown to be important regulators of the endocytic process. Recently, numerous interacting partners have been identified for each of these two classes of proteins. However, the details regarding the sequence of these interactions and the cross-talk between signaling pathways containing the different protein complexes are just beginning to be uncovered. In this review, we summarize these findings and discuss the role of GRKs and ß-arrestins, two families of key regulatory proteins that regulate G protein-coupled receptor endocytosis. [Copyright &y& Elsevier]

Dedicator-of-cytokinesis (DOCK) proteins are a family of guanine-nucleotide exchange factors (GEF) for Rho GTPases. The DOCK-D homology subfamily comprises DOCK9, DOCK10, and DOCK11. DOCK9 and DOCK11 are GEFs for Cdc42 and induce filopodia, while DOCK10 is a dual GEF for Cdc42 and Rac1 and induces filopodia and ruffles. We provide data showing that DOCK9, the only one of the DOCK-D members that is not considered hematopoietic, is nevertheless expressed at high levels in T lymphocytes, as do DOCK10 and DOCK11, although unlike these, it is not expressed in B lymphocytes. To investigate DOCK9 function, we have created a stable HeLa clone with inducible expression of HA-DOCK9. Induction of expression of HA-DOCK9 produced loss of elongation and polygonal shape of HeLa cells. Regarding membrane protrusions, HA-DOCK9 prominently induced filopodia, but also an increase of membrane ruffles. The latter was consistent with an increase in the levels of activation of Rac1, suggesting that DOCK9 carries a secondary ability to induce ruffles through activation of Rac1.