Role of Lbc RhoGEF in Gα12/13-induced signals to Rho GTPase

Abstract

Heterotrimeric Gα12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Gα signals to Rho. For example, p115 RhoGEF transduces Gα13 signals to Rho and inhibits Gα12/13 signals via the RhoGEF LH domain which links to Gα subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Gα12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Gα12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Gα12-induced RhoA activation in vivo. Complex formation between Gα12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Gα subunit signals. These findings support a role for Lbc in Gα12-induced signaling pathways to Rho.

Introduction

Heterotrimeric Gα12/13 signals induce cellular responses such as actin cytoskeletal changes and serum response element (SRE)-based gene transcription, and these responses have been found to occur via Rho small GTPase [1]. Rho is required for multiple cellular responses such as cell shape changes, contraction, retraction, migration and mitogenesis in most tissues [2]. Many of these Rho-dependent cellular responses are induced by stimulation of a subset of G protein-coupled receptors (GPCRs) [1]. Dysregulation of Rho pathway signals is implicated in several diseases [3], [4]. However, pathways by which Gα subunit signals activate Rho responses are partially understood.

GTPases such as Rho bind guanine nucleotides, and their activation state is determined by whether they bind GDP in the inactive state, or GTP in the active state. Rho guanine nucleotide exchange factors (GEFs) are key Rho regulators since they transduce extracellular signals to Rho and directly activate Rho GTPase by inducing rapid GDP/GTP exchange, resulting in GTP-bound Rho [5]. RhoGEFs have emerged as strong candidates for linking Gα subunit signals to Rho. Rho family GEFs contain a common DH (Dbl oncogene homology) domain which confers GEF activity, and this is usually followed by an adjacent PH (pleckstrin homology) domain [5]. In addition, RhoGEFs contain unique N- or C-terminal regions which represent regulatory regions. For example, p115 RhoGEF contains an N-terminal LH (Lsc homology) domain [6], [7], also known as the rgRGS (RhoGEF regulator of G protein signaling) domain [8]. The LH domain is required for the binding of activated Gα subunits to the RhoGEF. This binding can induce a conformational change, leading to RhoGEF stimulation [8]. For example, Gα13 can directly associate with, and activate p115 RhoGEF via the LH domain, leading to Rho activation [6]. Moreover, the LH domain can act as a GTPase activating protein (GAP) for activated Gα subunits, resulting in down-regulation of Gα signals [8], [9].

The Rho-specific Lbc RhoGEF [10], originally isolated as a transforming oncogene (onco-Lbc) [11], is a member of the RhoGEF family which includes p115 RhoGEF [12], its mouse homologue Lsc [13], p190 RhoGEF [14], PDZ RhoGEF [7] and LARG GEF [15]. While both onco-Lbc and its normal homologue proto-Lbc contain intact DH and PH domains, proto-Lbc contains an extended C-terminal region that is absent from onco-Lbc (ΔCT-Lbc) [16]. Subsequent reports of Lbc splice variants has highlighted the multi-functionality of the Lbc RhoGEF. For example, Brx is enriched in testes and implicated in nuclear hormone modulation [17], while AKAP-Lbc is enriched in the heart and functions as a protein kinase A-anchoring protein [18], [19].

Previous reports have suggested that Lbc RhoGEF may play a role in the Gα12/13 signaling pathway to Rho. For example, Lbc PH domain expression suppresses thrombin-induced astrocytoma cell rounding, a G12/13-linked response [20]. In addition, AKAP-Lbc cooperates with Gα12 to induce GTP-bound Rho formation in vivo [21], and Lbc is found physically associated with Gα12 [18], [21]. On this basis, here we have further investigated the signaling capacity of Lbc within the context of Gα12/13-induced signals.