IDENTIFICATION OF POTENTIAL TYROSINE-CONTAINING ENDOCYTIC MOTIFS IN THE CARBOXYL-TAIL AND 7TH TRANSMEMBRANE DOMAIN OF THE NEUROKININ-1 RECEPTOR

Although agonist-induced endocytosis of G-protein-coupled receptors is critical for receptor desensitization and resensitization, receptor m otifs that interact with the endocytic apparatus have not been adequat ely characterized, We examined the effects of mutating the rat neuroki nin-1 receptor on endocytosis using I-125-substance P, fluorescent sub stance P, and receptor antibodies, Substance P induced rapid internali zation of wildtype receptors that were targeted to perinuclear endosom es, Truncation of the C-tail at residues 324, 342, and 354 reduced int ernalization up to 60% and caused retention of receptors at the cell s urface and in superficial endosomes, Mutation of Tyr-341 and Tyr-349 i n potential tyrosine-containing endocytic motifs of the C-tail also im paired internalization. A Y305A mutant within the putative NPX(2-3)Y e ndocytic motif of the seventh transmembrane domain showed impaired sig naling and was minimally expressed at the plasma membrane but was foun d in cytoplasmic vesicles, In contrast, a Y305F mutant signaled normal ly and was normally expressed at the plasma membrane but showed impair ed internalization, Thus, endocytosis of the neurokinin 1 receptor rel ies on several tyrosine-containing sequences in the C-tail and seventh transmembrane domain.