Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including G(s), G(i), and G(q) subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT(4(a))) is coupled not only to heterotrimeric G(s) but also to G(13) protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT(4(a)) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5'-(3-O-thio)triphosphate binding to Galpha(13) protein. We then determined that expression of 5-HT(4(a)) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of Galpha(13) and RhoA. Finally, we have determined that expression of 5-HT(4(a)) receptor in neuroblastoma x glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT(4(a)) receptor-G(13) pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombininduced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.Microtubules, polymers of ␣-and ␤-tubulins, are key component of the cytoskeleton and are involved in multiple cellular processes such as migration, mitosis, protein, and organelle transport (1, 2). Microtubule dynamics and their spatial arrangements are affected by a number of signaling molecules. Conversely, changes in microtubule dynamics modulate intracellular signal transduction (for review, see Ref. 1).The actin cytoskeleton undergoes rearrangement under the control of various actin binding, capping, nucleating, and severing proteins, which are intimately involved in regulating the contractile status of the cells (3). Actin dynamics is regulated via transduction of extracellular signals to intracellular events primarily through the members of the Rho family of small GTPases. Rho is known to induce stress fiber formation, whereas Cdc42 and Rac are involved in formation of lamellipodia and filopodia, respectively (4).Microtubule disassembly promotes the formation of actin stress fibers and enhances the contractility of cells (5). Agents such as nocodazole or vinblastine that disrupt microtubules induce rapid assembly of actin filaments and focal adhesions (6), whereas microtubule stabilization with taxol attenuated these effects. Regulation of the actin cytoskeleton by microtubules requires the Rho GTPases (for review, see Ref. 7). However, the mechanisms involved in the coordinated regulation of microtubules and the actin cytoskeleton remain poorly understood. LIMK1 1 is a serine/threonine kinase that regulates a...

Abstract-Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of G␤␥ subunits, transducin. We have determined that G␤␥ subunits but not G␣ subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that G␤␥ subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, G␤␥ subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that G␤␥ subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production. Key Words: Rho GTPases Ⅲ guanine nucleotide exchange factor Ⅲ actin cytoskeleton Ⅲ serum response element Ⅲ reactive oxygen species G uanine nucleotide exchange factors of the Dbl family are multifunctional proteins that transduce diverse intracellular signals leading to the activation of Rho GTPases (see review 1 ). The tandem Dbl homology (DH) and pleckstrin homology (PH) domains are shared by all members of this family and represent a structural module responsible for the catalyzing the GDP-GTP exchange of Rho proteins and, therefore, their activation. Previous studies have shown that the DH domain is responsible for the catalytic core of the RhoGEF enzymatic activity, 2 whereas the PH domain is involved in intracellular targeting, lipid-binding, and protein interaction. 1,3 Furthermore, guanine nucleotide exchange factors (GEFs) may contain other conserved motifs such as RGS (regulator of G-protein signaling) motif, SH3 (Src homology) motif, and proline-rich SH3-binding motif, 1 thereby supplying RhoGEFs with additional signaling functions.Diverse upstream signals that stimulate RhoGEFs catalytic activity include heterotrimeric G proteins, protein kinases, adapto...

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified ␣SNAP as an interactive partner of G␣ 12 using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of ␣SNAP with G␣ 12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G␣ 12 (1-37 amino acids) was necessary and sufficient for its interaction with ␣SNAP. G␣ 13 with its N-terminal extension replaced by that of G␣ 12 acquired the ability to bind to ␣SNAP, whereas G␣ 12 with its N terminus replaced by that of G␣ 13 lost this ability. Using four point mutants of ␣SNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of ␣SNAP was involved in its interaction with G␣ 12 . Co-transfection of human umbilical vein endothelial cells with G␣ 12 and ␣SNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of ␣SNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G␣ 12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the ␣ subunit of G 12 and ␣SNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.