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.

Epitope and fluorescent tagging of trimeric G protein α subunits poses challenges due to potential disruption of functional regions, including the receptor‐interacting N‐ and C‐termini. In previous work, we inserted green fluorescent protein (GFP) and a Myc tag into a specific loop within the helical domain of Gα12, flanking each adduct with the sextet ser‐gly‐gly‐gly‐gly‐ser, and showed these tagged Gα12 variants to retain interaction with downstream target proteins, stimulate serum response factor, and exhibit conformational activation. Recently, we employed a similar approach for tagging Gα13, which shares 67% identity with Gα12 but has significant sequence divergence in the helical domain. Using molecular visualization software (PyMol) to superimpose the crystal structures of Gα12 and Gα13, we identified Gα13 residues corresponding to the site of tag insertion in Gα12, and made key substitutions of Gα12‐specific residues into Gα13. GFP‐tagged Gα13 is detectable in transfected cells by epifluorescence microscopy and immunoblotting, and shows robust activation of serum response factor. We are utilizing tagged Gα13 in binding assays to determine whether mutations in Gα13 disrupt binding to effectors. Furthermore, these tagged constructs are facilitating side‐by‐side comparisons of Gα12 and Gα13 in their relative affinity for common downstream effector proteins, such as Rho‐specific guanine nucleotide exchange factors, cadherins, and other targets. We acknowledge support from the North Carolina Biotechnology Center (DNB) and the Lineberger Comprehensive Cancer Center (TEM).

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The Rho family of small GTP‐binding proteins are key regulators of cytoskeletal dynamics and cell growth. Rho activation is triggered by guanine nucleotide exchange factors (RhoGEFs), and a specific subset of these proteins, including leukemia‐associated RhoGEF (LARG), are stimulated by trimeric G protein α subunits of the G12/13 class. This interaction is mediated by a regulator of G protein signaling (RGS) homology domain common to these RhoGEFs; however, a RhoGEF lacking an RGS homology domain, termed AKAP‐Lbc, is also activated by the G12/13 class in a signaling pathway required in cardiac development. To identify AKAP‐Lbc regions involved in Gα12 binding, we compared AKAP‐Lbc to other Gα12 targets and found a C‐terminal region with weak homology to axin. A GST‐fusion of this region co‐precipitated Gα12 in constitutively active and inactive forms, in contrast to the activation‐dependent interaction of Gα12 with LARG. In addition, binding experiments utilizing Gα12 and Gα13 harboring a common epitope tag revealed this AKAP‐Lbc domain as preferential for Gα12 interaction, whereas the RGS homology domain of LARG bound Gα13 with greater affinity than Gα12. These findings reveal a novel, Gα12‐specific mechanism for engagement of a non‐RGS RhoGEF by the G12/13 class of trimeric G proteins. Grant Funding Source: Supported by the GlaxoSmithKline Foundation and Lineberger Comprehensive Cancer Center