Abstract

The histamine H₁ receptor (H1R)-mediated signaling cascade is inhibited by phorbol ester-induced protein kinase C (PKC) activation. Cloning studies of the H1Rs have shown that several potential PKC phosphorylation sites are located in the third intracellular loop of H1R. To elucidate the molecular mechanism of PKC-mediated desensitization, we identified amino acid residues that are involved in the desensitization of the H1R. Two amino acid residues (Ser³⁹⁶, Ser³⁹⁸) were determined to be PKC phosphorylation sites by in vitro phosphorylation studies using a series of synthetic peptides. Treatment with phorbol ester decreased histamine-induced accumulation of inositol phosphates in Chinese hamster ovary cells expressing the H1R with a rightward shift in the EC₅₀ value, which implies the uncoupling of the receptor from the G protein. Site-directed mutagenesis studies showed that substitution of alanine for Ser³⁹⁸ but not for Ser³⁹⁶ markedly attenuated the effect of phorbol ester, which suggests that the Ser³⁹⁸ residue was primarily involved in PKC-mediated desensitization.