Distinct Pathways of ERK1/2 Activation by Hydroxy-Carboxylic Acid Receptor-1

Mechanistic investigations have shown that, upon agonist activation, hydroxy-carboxylic acid receptor-1(HCA(1)) couples to a G(i) protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for HCA(1) signaling remain largely unknown. Using CHO-K1 cells stably expressing HCA(1), and L6 cells, which endogenously express rat HCA(1) receptors, we found that activation of ERK1/2 by HCA(1) was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that HCA(1) induced ERK1/2 activation via the extracellular Ca(2+), PKC and IGF-I receptor transactivation-dependent pathways. In addition, we observed that pretreated the cells with M119K, an inhibitor of G(βγ) subunit-dependent signaling, effectively attenuated the ERK1/2 activation triggered by HCA(1), suggesting a critical role for βγ-subunits in HCA(1)-activated ERK1/2 phosphorylation. Furthermore, the present results also indicated that the arrestin2/3 were not required for ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to agonist, HCA(1) receptors initially activate G(i), leading to dissociation of the G(βγ) subunit from activated G(i), and subsequently induce ERK1/2 activation via two distinct pathways: one PKC-dependent pathway and the other IGF-IR transactivation-dependent pathway. Our results provide the first in-depth evidence that defines the molecular mechanism of HCA(1)-mediated ERK1/2 activation.