Free fatty acid receptor 4 activation induces lysophosphatidic acid receptor 1 (LPA1) desensitization independent of LPA1 internalization and heterodimerization

A. Meizoso-Huesca,^1,2 S. Villegas-Comonfort,¹ T. Romero-Avila¹ and A. Garcia-Sainz,¹ ¹Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, C.U., Av. Universidad Nº 3000, 04510 México City, México and ²School of Biomedical Sciences, The University of Queensland, St, Lucia, QLD 4072, Australia.

Lysophosphatidic acid is one of the main mitogenic compounds of blood serum. Its functions are mediated by a family of 6 GPCRs (known as LPA_1-6) expressed differentially among the tissues of the body. On the other hand, the Free Fatty Acids Receptor 4 (FFA4) is a well-known GPCR of long chain free fatty acids that has been related to anti-diabetic and anti-inflammatory processes as well as brain development.

In different cancer types (prostate, breast, and ovary), FFA4 activation has been proven to block mitogenic features of LPA₁ activity. In consequence, the crosstalk between these receptors is of pathophysiological relevance.

The aim of this study was to explore the crosstalk between LPA and FFA4 employing co-expression of fluorescent protein-tagged receptors on HEK 293 cells. Functional FFA4-mediated LPA₁ desensitization was assessed by calcium fluorometry of cells in suspension and western blotting. As a classic negative-regulation modification, phosphorylation of receptors in response to their agonists was tested: FFA4 activation induced phosphorylation of both receptors. As expected, LPA₁ activation induced phosphorylation of LPA₁, but not of FFA4. LPA₁ activation drove internalization of both receptors into heterogeneous types of vesicles. FFA4 agonist led to internalization of FFA4 but not of LPA₁, suggesting a desensitization mechanism independent of the internalization of the receptor. Dimerization of GPCRs in response to a stimulus has been shown to induce modifications in pharmacological properties of these proteins, leading to changes of the affinity for their ligands. In order to test this, fatty acid-induced FFA4-LPA₁ interaction was observed using Forster Resonance Energy Transfer (FRET) and co-immunoprecipitation; however, such interaction took place once the desensitization was already established. Data indicate that FFA4 activation induces LPA₁ desensitization in an internalization-independent mechanism and that during the first moments of this desensitization, heterodimerization does not play a relevant role.