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Center for Basic Research in Digestive Diseases and the Department
of Biochemistry and Molecular Biology, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
Receptor phosphorylation has been implicated in desensitization
responses to some agonist ligands, in which receptors may become
uncoupled from G proteins and move into cellular compartments inaccessible to hydrophilic ligands. Understanding of the linkage between these processes, however, has come largely from recombinant receptor-bearing cell systems with consensus sites of kinase action mutagenized. We recently established methodology permitting direct assessment of sites of phosphorylation of the cholecystokinin receptor
(CCKR) in its native milieu in the pancreatic acinar cell and in a
Chinese hamster ovary (CHO)-CCKR cell line (1, 2). Although CCK
binding leads to phosphorylation of serine residues within the third
intracellular loop of the receptor in both cell types, there are clear
differences in the time course of phosphorylation, in the balance of
action of kinases and a receptor phosphatase, and in a few of the
distinct sites phosphorylated. In this work, we have directly assessed
the inositol 1,4,5-triphosphate responses to CCK and desensitization of
these responses in both cells. CHO cell lines expressing receptor
mutants with protein kinase C consensus sites modified were also
studied. CCK-stimulated inositol 1,4,5-triphosphate responses in both
cells expressing wild-type receptors were rapidly and completely
desensitized, associated with the onset of receptor phosphorylation.
However, despite maintenance of the phosphorylated state of the
receptor in the CHO-CCKR cell and its dephosphorylation returning the
receptor to its basal state in the acinar cell, desensitization
continued to be present in both. Mutagenesis of Ser260 and Ser264 to
alanines individually reduced receptor phosphorylation by approximately
50%, whereas the dual mutant completely eliminated agonist-stimulated
phosphorylation. Because other sites of phosphorylation were still
intact in this construct, this raises the possibility of hierarchical
phosphorylation with these two sites key in making other sites
accessible to kinases. Constructs modifying Ser264 delayed the onset of
desensitization, whereas all constructs proceeded to achieve complete
desensitization by 10 min. Receptor internalization occurred
independent of its phosphorylation state in the CHO cell lines,
explaining the desensitization observed. In the acinar cell in which
the receptor remains on the cell surface after agonist occupation, we
postulate that receptor insulation achieves similar uncoupling from G
protein association as is achieved by receptor phosphorylation early
after agonist occupation.
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