Desensitization of G-protein-coupled receptors: Agonist-induced phosphorylation of the chemoattractant receptor cAR1 lowers its intrinsic affinity for cAMP

Agonist-induced phosphorylation of G-protein-coupled receptors has been shown to facilitate the desensitization processes, such as receptor internalization, decreased efficiency of coupling to G-proteins, or decreased ligand affinity. The lowered affinity may be an intrinsic property of the phosphorylated receptor or it may be the result of altered interactions between the modified receptor and downstream components such as G-proteins or arrestins. To address this issue, we purified cAR1, the major chemoattractant receptor of Dictyostelium discoideum by a strategy that is independent of the ligand binding capacity of the receptor. To our knowledge, this represents the first successful purification of a chemoattractant receptor. The hexyl- histidine-tagged receptor was solubilized from a highly enriched plasma membrane preparation and purified by Ni²⁺- chelating chromatography. The protocol offers a simple way to purify 100-500 μg of a G-protein coupled receptor that can be targeted to the plasma membrane of D. discoideum. The K(d) value for the purified cAR1 was about 200 nM, consistent with that of receptors that are not coupled to G-proteins in intact cells. In contrast, the affinity of phosphorylated cAR1, purified from desensitized cells, was about three times lower. Treatment of the phosphorylated receptor with protein phosphatases caused dephosphorylation and parallel restoration of higher affinity. We propose that ligand-induced phosphorylation of G-protein- coupled receptors causes a decrease in intrinsic affinity and may be useful in maintaining the receptor's sensitivity at high agonist levels. This affinity decrease may precede other processes such as receptor internalization or uncoupling from G-proteins.