ROLES OF CHOLECYSTOKININ RECEPTOR PHOSPHORYLATION IN AGONIST-STIMULATED DESENSITIZATION OF PANCREATIC ACINAR-CELLS AND RECEPTOR-BEARING CHINESE-HAMSTER OVARY CHOLECYSTOKININ RECEPTOR-CELLS

Receptor phosphorylation has been implicated in desensitization respon ses to some agonist ligands, in which receptors may become uncoupled f rom G proteins and move into cellular compartments inaccessible to hyd rophilic ligands. Understanding of the linkage between these processes , however, has come largely from recombinant receptor-bearing cell sys tems with consensus sites of kinase action mutagenized. We recently es tablished methodology permitting direct assessment of sites of phospho rylation of the cholecystokinin receptor (CCKR) in its native milieu i n the pancreatic acinar cell and in a Chinese hamster ovary (CHO)-CCKR cell line (1, 2). Although CCK binding leads to phosphorylation of se rine residues within the third intracellular loop of the receptor in b oth cell types, there are clear differences in the time course of phos phorylation, in the balance of action of kinases and a receptor phosph atase, and in a few of the distinct sites phosphorylated. In this work , we have directly assessed the inositol 1,4,5-triphos phate responses to CCK and desensitization of these responses in both cells. CHO cell lines expressing receptor mutants with protein kinase C consensus sit es modified were also studied. CCK-stimulated inositol 1,4,5-triphosph ate responses in both cells expressing wild-type receptors were rapidl y and completely desensitized, associated with the onset of receptor p hosphorylation. However, despite maintenance of the phosphorylated sta te of the receptor in the CHO-CCKR cell and its dephosphorylation retu rning the receptor to its basal state in the acinar cell, desensitizat ion continued to be present in both. Mutagenesis of Ser260 and Ser264 to alanines individually reduced receptor phosphorylation by approxima tely 50%, whereas the dual mutant completely eliminated agonist-stimul ated phosphorylation. Because other sites of phosphorylation were stil l intact in this construct, this raises the possibility of hierarchica l phosphorylation with these two sites key in making other sites acces sible to kinases. Constructs modifying Ser264 delayed the onset of des ensitization, whereas all constructs proceeded to achieve complete des ensitization by 10 min. Receptor internalization occurred independent of its phosphorylation state in the CHO cell lines, explaining the des ensitization observed. In the acinar cell in which the receptor remain s on the cell surface after agonist occupation, we postulate that rece ptor insulation achieves similar uncoupling from G protein association as is achieved by receptor phosphorylation early after agonist occupa tion.