Abstract
AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. We identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that we also discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. Our results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs.
Highlights
Cells respond to changes in their environment through complex networks of intracellular signaling proteins
A-kinase anchoring proteins (AKAP)-Lbc was discovered by Diviani, Scott and colleagues as a Rho-activating guanine nucleotide exchange factor (RhoGEF) stimulated by Gα12 binding to drive Rho activation [12]
Together with the report of Gα13-Rgnef interaction, our results suggest this region of homology in AKAP-Lbc, p114RhoGEF and Rgnef is derived from a G12/13 binding motif in an ancestral RhoGEF, but became specialized for Gα13 binding in Rgnef and Gα12 binding in a more recent common ancestor of AKAP-Lbc and p114RhoGEF
Summary
Cells respond to changes in their environment through complex networks of intracellular signaling proteins. Many G12/13 mediated responses require downstream activation of Rho, a small GTPase that, when activated by exchange of bound GDP for GTP, drives signaling through effector proteins that include mDia and Rho-associated kinase [2]. The classical link between the G12/13 subfamily and Rho is a small subclass of Rho-directed guanine nucleotide exchange factors (RhoGEFs) comprising p115RhoGEF, leukemia-associated RhoGEF (LARG), and PDZ-RhoGEF. These proteins are bound directly by GTP-bound a subunits of the G12/13 subfamily, triggering the RhoGEF to bind and activate Rho [3]. A common feature of these G12/13-responsive RhoGEFs is an RGS-homology (RH) domain, similar in sequence to a hallmark structural region in regulators of G protein signaling (RGS) that bind activated a subunits and
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