MULTIPLE PHOSPHORYLATION SITES ARE REQUIRED FOR PATHWAY-SELECTIVE UNCOUPLING OF THE 5-HYDROXYTRYPTAMINE(1A) RECEPTOR BY PROTEIN-KINASE-C

Classically, acute uncoupling of G protein-linked receptors has been p resented as a nonselective process in which modification of the recept or by phosphorylation leads to reduction in or the loss of coupling to all effecters. Investigation of multiple signaling pathways has modif ied this view: for example, when expressed in Ltk(-) fibroblasts, the 5-hydroxytryptamine(1A) (5-HT1A) receptor couples to both stimulation of intracellular calcium mobilization (via inositol phosphate generati on) and inhibition of cAMP accumulation. Acute pretreatment for 2 min with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator o f protein kinase C (PKC), abolished the intracellular calcium response induced by 100 nM 5-HT but did not reduce 5-HT1A receptor-mediated in hibition of cAMP. In the present report, mutant 5-HT1A receptors lacki ng one, two, or three putative PKC phosphorylation sites located in th e receptor's third cytoplasmic loop were constructed and transfected s eparately into Ltk(-) cells. The receptor mutants displayed agonist af finities similar to that of the nonmutated receptor. The relative magn itudes of 5-HT-induced changes in intracellular calcium or forskolin-s timulated cAMP levels were also similar to those of the wild-type rece ptor for all except one of the mutants. In addition, TPA treatment did not change the magnitude or potency of 5-HT-induced inhibition of pro staglandin E(1)-stimulated cAMP accumulation in the wild-type or tripl e-mutant 5-HT1A receptor clones. On pretreatment with TPA, there was a progressive recovery to 74% of the control 5-HT-induced increase in c alcium mobilization as PKC sites were eliminated from the receptor, in dicating that multiple phosphorylation sites are required for PKC-medi ated receptor uncoupling. Also, these results indicate that receptor p hosphorylation selectively uncouples the 5-HT1A receptor from enhancem ent of calcium mobilization without reducing receptor-mediated inhibit ion of cAMP. Thus, phosphorylation by PKC can sculpt receptor signalin g via pathway-selective uncoupling.