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 sho wn to facilitate the desensitization processes, such as receptor internaliz ation, decreased efficiency of coupling to G-proteins, or decreased ligand affinity. The lowered affinity may be an intrinsic property of the phosphor ylated receptor or it may be the result of altered interactions between the modified receptor and downstream components such as G-proteins or arrestin s, To address this issue, we puri fled cAR1, the major chemoattractant rece ptor of Dictyostelium discoideum by a strategy that is independent of the l igand binding capacity-of the receptor. To our knowledge, this represents t he first successful purification of a chemoattractant receptor. The hexyl-h istidine-tagged receptor was solubilized from a highly enriched plasma memb rane preparation and purified by Ni2+-chelating chromatography, The protoco l offers a simple way to purify 100-500 mu g of a G-protein coupled recepto r that can be targeted to the plasma membrane of D. discoideum. The K-d val ue for the purified cAR1 was about 200 nM, consistent with that of receptor s that are not coupled to G-proteins in intact cells. In contrast, the affi nity of phosphorylated cAR1, purified from desensitized cells, was about th ree times lower. Treatment of the phosphorylated receptor with protein phos phatases caused dephosphorylation and parallel restoration of higher affini ty. We propose that ligand-induced phosphorylation of G-protein-coupled rec eptors causes decrease in intrinsic affinity and may be useful in maintaini ng the receptor's sensitivity at high agonist levels. This affinity decreas e may precede other processes such as receptor internalization or uncouplin g from G-proteins.