Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.
Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59-62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24-98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 +/- 0.15 nM) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of 125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 +/- 1.4, 0.85-1.6, and 0.7 +/- 0.2 nM respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.
Berkhout
T A
TA
Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, United Kingdom.
Sarau
H M
HM
Moores
K
K
White
J R
JR
Elshourbagy
N
N
Appelbaum
E
E
Reape
R J
RJ
Brawner
M
M
Makwana
J
J
Foley
J J
JJ
Schmidt
D B
DB
Imburgia
C
C
McNulty
D
D
Matthews
J
J
O'Donnell
K
K
O'Shannessy
D
D
Scott
M
M
Groot
P H
PH
Macphee
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001161:Arteriosclerosis / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
C099677:CCL13 protein, human; C515334:CCR2 protein, human; D018945:Monocyte Chemoattractant Proteins; D012333:RNA, Messenger; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D002118:Calcium