Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

Abstract

ResultsThe lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.ConclusionOur data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

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

“…Stimulation with 1 lM LPA for 5 min increased LPA 1 phosphorylation (Fig. 3, panel D), in agreement with previous reports [26,41] but no increase in FFA4 phosphorylation was observed under these conditions (Fig. 3, panel C).…”

Lysophosphatidic acid receptor LPA3 prevents oxidative stress and cellular senescence in Hutchinson–Gilford progeria syndrome

“…First, measuring the mRNA stability of Lpar3 showed no differences between Lamin A and Progerin cells (Figure B). Notably, having demonstrated the constitutive internalization of GPCR over time, Alcántara‐Hernández et al showed that these receptors will reach the lysosomal compartment for degradation (Alcántara‐Hernández, Hernández‐Méndez, Campos‐Martínez, Meizoso‐Huesca, & García‐Sáinz, ). Treatment with 20 mM of NH 4 Cl and 20 nM of Bafilomycin A1, but not with 5 μM of MG132, rescued LPA 3 degradation in Progerin HEK293 cells (Figure a–b).…”

“…Although LPA 1 and LPA 2 both belong to the EDG protein family like LPA 3 , the protein levels of LPA 1 and LPA 2 are not consistently observed to be downregulated by lysosomal pathways in different senescent cells. A previous study showed that LPA 3 is mostly phosphorylated and internalized after induction, but LPA 1 and LPA 2 require a longer time and a larger induction to induce internalization (Alcántara‐Hernández et al, ). It has been suggested that PDZ domains on the C‐terminal of LPA 1 and LPA 2 might be responsible for prolonged cell surface resident time and reduced rates of internalization (Marchese, Paing, Temple, & Trejo, ).…”

Endogenous lysophosphatidic acid (LPA1) receptor agonists demonstrate ligand bias between calcium and ERK signalling pathways in human lung fibroblasts

“…This occurred between 2 and 3 min for the calcium response and at 5 min for the pERK response. Recent data have demonstrated that LPA 1 receptor phosphorylation occurs after 15–30 min treatment with LPA, with internalization occurring after 30–60 min (Alcántara‐Hernández et al, ). Although we cannot completely rule out a contribution of these processes, this suggests that the responses we are measuring will not be affected by differences in receptor state but are in fact due to differential activation of the pathways being measured.…”