Volume 272, Number 4,
Issue of January 24, 1997
pp. 2363-2372
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Potential Tyrosine-containing Endocytic
Motifs in the Carboxyl-tail and Seventh Transmembrane Domain of
the Neurokinin 1 Receptor
(Received for publication, March 22, 1996, and in revised form, November 1, 1996)
Stephan K.
Böhm
,
Lev M.
Khitin
,
Steven P.
Smeekens
¶
,
Eileen F.
Grady
,
Donald G.
Payan
¶
and
Nigel W.
Bunnett
**
From the Departments of 
Surgery, 
Medicine and
** Physiology, University of California,
San Francisco, California 94143 and ¶ Khepri Pharmaceuticals
Inc., South San Francisco, California 94080
Although agonist-induced endocytosis of
G-protein-coupled receptors is critical for receptor desensitization
and resensitization, receptor motifs that interact with the endocytic
apparatus have not been adequately characterized. We examined the
effects of mutating the rat neurokinin-1 receptor on endocytosis using
125I-substance P, fluorescent substance P, and receptor
antibodies. Substance P induced rapid internalization of wild-type
receptors that were targeted to perinuclear endosomes. Truncation of
the C-tail at residues 324, 342, and 354 reduced internalization up to
60% and caused retention of receptors at the cell surface and in
superficial endosomes. Mutation of Tyr-341 and Tyr-349 in potential tyrosine-containing endocytic motifs of the C-tail also impaired internalization. A Y305A mutant within the putative
NPX2-3Y endocytic motif of the seventh
transmembrane domain showed impaired signaling and was minimally
expressed at the plasma membrane but was found in cytoplasmic vesicles.
In contrast, a Y305F mutant signaled normally and was normally
expressed at the plasma membrane but showed impaired internalization.
Thus, endocytosis of the neurokinin 1 receptor relies on several
tyrosine-containing sequences in the C-tail and seventh transmembrane
domain.
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