Structure/function relationships of CCR8 agonists and antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist.
We describe here the interactions of CCR8 with its ligands using both CCR8 transfectants and a T-cell line expressing the receptor endogenously. Of the CCR8 agonists reported previously, only CCL1 and vMIP-I exhibited potency in assays of intracellular calcium flux, chemotaxis, and receptor internalization, this latter mechanism being dependent upon the expression of beta-arrestins 1 and 2 but independent of Galpha(i) signaling. NH(2)-terminal extension of the mature CCL1 sequence by a serine residue (Ser-CCL1) resulted in a partial agonist with a reduced affinity for CCR8, suggesting that the NH(2) terminus of the ligand plays a role in ligand binding to an intrahelical site. Attempts to identify key residues within this site revealed that the conserved glutamic acid residue in transmembrane helix 7, Glu-286, is crucial for trafficking of the receptor to the cell surface, while Asp-97 of transmembrane helix 2 is dispensable. CCL7 was found to inhibit both Ser-CCL1 and vMIP-I responses but not those of CCL1 itself. Similarly, vMIP-I responses were more than 2 orders of magnitude more sensitive to the specific CCR8 antagonist MC148 than those induced by CCL1, which is difficult to reconcile with the reported affinities for the receptor. Collectively, these data suggest that the CCR8 ligands are allotropic, binding to distinct sites within CCR8 and that the human immune system may have evolved to use CCL7 as a selective antagonist of viral chemokine activity at CCR8 but not those of the host ligand.
Fox
James M
JM
National Heart and Lung Institute Division, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
Najarro
Pilar
P
Smith
Geoffrey L
GL
Struyf
Sofie
S
Proost
Paul
P
Pease
James E
JE
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D054402:Chemokine CCL1; D054407:Chemokine CCL4; D019742:Chemokines, CC / Q000737:chemistry / Q000494:pharmacology; D002633:Chemotaxis; D018698:Glutamic Acid / Q000737:chemistry; D008024:Ligands; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054401:Receptors, CCR8; D019707:Receptors, Chemokine / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014764:Viral Proteins
C065971:CCL1 protein, human; C103330:CCR8 protein, human; C489319:Ccr8 protein, mouse; D054402:Chemokine CCL1; D054407:Chemokine CCL4; D019742:Chemokines, CC; D008024:Ligands; C435144:MC148 protein, poxvirus molluscum contagiosum; D019402:Macrophage Inflammatory Proteins; D054401:Receptors, CCR8; D019707:Receptors, Chemokine; D014764:Viral Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D002118:Calcium