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(Received for publication, April 30, 1996, and in revised form, January 21, 1997)
,
,
and¶
From the We have characterized the ligand-enhanced
phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of
clonal 3ASubE cell lines expressing receptors truncated or mutated in
the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a
stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss
of melanoma growth stimulatory activity/growth-related protein
(MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained
based upon altered ligand binding affinity or receptor number. 3ASubE
cells expressing 342T or CXCR2 with mutation of Ser-342, -346, -347, and -348 to alanine (4A) exhibit strong mobilization of
Ca2+ in response to ligand (interleukin-8 or
MGSA/GRO), with a recovery phase significantly slower than that of
cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2,
which is 93% desensitized by 20 nM ligand, the 331T, 342T,
and 4A CXCR2 mutants do not undergo significant ligand-induced
desensitization, and respond to a second ligand challenge by mobilizing
Ca2+. The 3ASubE cells expressing CXCR2 with mutation of
Ser-346, -347, and -348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and
are 60-70% desensitized following the initial ligand challenge. WT
CXCR2 phosphorylation and desensitization occur in <1 min, while
receptor sequestration is a much later event (30-60 min). However,
mutant receptors that are neither phosphorylated nor desensitized in
response to ligand are <10% sequestered 60 min following ligand
challenge. These data demonstrate for the first time that ligand
binding to CXCR2 results in receptor phosphorylation, desensitization,
and sequestration and that serine residues 342 and 346-348 participate
in the desensitization and sequestration processes.
Department of Cell Biology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-2175, the
¶ Veterans Affairs Medical Center, Nashville, Tennessee
37212-2637, and § SmithKline Beecham Pharmaceuticals,
Department of Immunology,
King of Prussia, Pennsylvania 19406-0939
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