Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and ß-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1.

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gßγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gßγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.

G(12)/G(13)-mediated signalling in mammalian physiology and disease.

The human genome encodes hundreds of G-protein-coupled receptors. Their intracellular effects, however, are mediated by only four families of heterotrimeric G proteins: G(s), G(i)/G(o), G(q)/G(11) and G(12)/G(13). Progress in the knowledge about the G(12)/G(13) family has somewhat lagged behind because their downstream effectors remained unknown for several years, and tools to specifically interfere with G(12)/G(13)-mediated signalling were, therefore, missing. However, with the identification of G(12)/G(13)-regulated signalling pathways and the recent application of new techniques, such as conditional gene inactivation, RNA interference or expression of inhibitory proteins, new insights into the in vivo functions of this G-protein family have been gained. It has become clear that this pathway regulates cellular proliferation, movement and morphology in many different organs and that it is centrally involved in various diseases including cancer and cardiovascular disorders. Here, we focus on recent progress made in the analyses of the in vivo functions of mammalian G(12)/G(13)-mediated signalling.

Mitogenic signaling by lysophosphatidic acid (LPA) involves Galpha12.

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as G(i), G(q), and G12/13. Previous studies have shown that the overexpression of wild-type Galpha12 (Galpha12WT) results in the oncogenic transformation of NIH3T3 cells (Galpha12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Galpha12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Galpha12WT-NIH3T3 with 2 muM LPA leads to the activation of Galpha12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Galpha12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Galpha12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Galpha12, but not the closely related Galpha13. Pretreatment of Galpha12WT-NIH3T3 cells with suramin (100 microM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Galpha12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Galpha12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.