Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2.
The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.
Jarnagin
K
K
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA. kjarnagin@iconixpharm.com
Grunberger
D
D
Mulkins
M
M
Wong
B
B
Hemmerich
S
S
Paavola
C
C
Bloom
A
A
Bhakta
S
S
Diehl
F
F
Freedman
R
R
McCarley
D
D
Polsky
I
I
Ping-Tsou
A
A
Kosaka
A
A
Handel
T M
TM
D000596:Amino Acids / Q000302:isolation & purification / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000502:physiology; D002464:Cell Migration Inhibition; D018932:Chemokine CCL2 / Q000819:agonists / Q000235:genetics / Q000502:physiology*; D000242:Cyclic AMP / Q000037:antagonists & inhibitors; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000378:metabolism / Q000502:physiology*; D018121:Receptors, Cytokine / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000502:physiology
D000596:Amino Acids; C515334:CCR2 protein, human; D018932:Chemokine CCL2; D010446:Peptide Fragments; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D014443:Tyrosine; D000242:Cyclic AMP; D002118:Calcium