Identification and characterization of a lysophosphatidic acid receptor

Abstract

A specific binding site for 1-[3H]stearoyl-lysophosphatidic acid (stearoyl-LPA) was identified and characterized in membranes prepared from rat brain and Swiss 3T3 fibroblasts. Specific binding of [3H]LPA to these sites was protein dependent, was saturable, reached equilibrium in 15 min, and was displacable by the addition of excess unlabeled LPA. Scatchard analysis of saturation binding experiments indicated that these sites had affinities of 2.0 +/- 0.5 nM and 5.4 +/- 2.6 nM and densities of 19 +/- 3 fmol/micrograms of protein and 38 +/- 6 fmol/micrograms of protein in rat brain and 3T3 cell membranes, respectively. Various LPAs, with different acyl groups in the sn-1-position, competed with [3H]LPA for these binding sites, with a rank order of potency of 1-oleoyl-LPA > 1-stearoyl-LPA = 1-palmitoyl-LPA > 1-myristoyl-LPA. Phosphatidic acid also bound to these sites, but with lower affinity than any LPA tested. Neither lysophosphatidylcholine, lysophosphatidylethanolamine, nor any free fatty acid competed with [3H]LPA for these binding sites. Binding of [3H]LPA to these sites was regulated by nonhydrolyzable guanine nucleotides in both rat brain and 3T3 cell membranes. Furthermore, in 3T3 cells, these sites were regulated by cell density. It was subsequently determined that LPA induced a transient increase in intracellular Ca2+ levels in 3T3 cells. The concentrations required for this response, as well as the rank order of potency of the various LPAs and phosphatidic acid, correlated with the affinity of these compounds for the [3H]LPA binding site. These results suggest that the specific, high affinity, binding sites for [3H]LPA are G protein-coupled receptors.