ORGANIZATION AND DIFFERENTIAL EXPRESSION OF THE HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN-1 RECEPTOR GENE - EVIDENCE FOR THE ROLE OF THE CARBOXYL-TERMINAL TAIL IN RECEPTOR TRAFFICKING
Lm. Wong et al., ORGANIZATION AND DIFFERENTIAL EXPRESSION OF THE HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN-1 RECEPTOR GENE - EVIDENCE FOR THE ROLE OF THE CARBOXYL-TERMINAL TAIL IN RECEPTOR TRAFFICKING, The Journal of biological chemistry, 272(2), 1997, pp. 1038-1045
Two forms of the monocyte chemoattractant protein-1 receptors (the typ
e A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the
type B MCP-1 receptor (CCR-2B) have been recently cloned and found to
differ only in their terminal carboxyl tails. Here, we report that th
e two isoforms are alternatively spliced variants of a single MCP-1 re
ceptor gene, Sequencing of the gene revealed that the 47-amino acid ca
rboxyl tail of CCR2B was located in the same exon as the seven transme
mbrane domains of the receptor, and the 61-amino acid tail of CCR2A wa
s in a downstream exon. Examination of freshly isolated human monocyte
s by reverse transcriptase polymerase chain reaction revealed that CCR
2B was the predominant isoform and that message levels of both CCR2A a
nd CCR2B decreased as the monocytes differentiated into macrophages. I
n stably transfected cell lines, CCR2B trafficked well to the cell sur
face, but CCR2A was found predominately in the cytoplasm. Equilibrium
binding studies revealed that those CCR2A receptors that successfully
trafficked to the cell surface bound MCP-1 with high affinity (K-d = 3
10 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B m
ediated agonist-dependent calcium mobilization, as well as inhibition
of adenylyl cyclase. Creation of chimeras between CCR2A and the human
thrombin receptor revealed that the cytoplasmic retention of CCR2A was
due to its terminal carboxyl tail. Progressive truncation of the carb
oxyl tail indicated that a cytoplasmic retention signal(s) was located
between residues 316 and 349. These data indicate that the alternativ
ely spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with
high affinity and is a functional receptor and that expression at the
cell surface is controlled by amino acid sequences located in the term
inal carboxyl tail.