Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528 - Implications for receptor biological responses

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-acti vated protein kinase activity coupled to the spontaneous autoactivated peri pheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line st ably transfected with human CB2, In the present report, we studied the effe ct of SR 144528 on CB2 phosphorylation, The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-termina l CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352, We first showed that CB2 is constitutively acti ve, phosphorylated, and internalized at the basal level. By blocking autoac tivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitog en-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated an d maintained in this phosphorylated state for more than 8 h after agonist t reatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144 528, paradoxically leading to a nonphosphorylated CB2 which could then be f ully activated by CP-55,940, The process of CP-55,940-induced receptor phos phorylation followed by SR 144528 induced receptor dephosphorylation kept r ecurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finall y, we showed that autophosphorylation and CP-55,940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use G(i)b eta(gamma), In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.