Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region

Abstract

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.

Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10

“…Sulfated tyrosines at the N-termini of chemokine receptors play a critical role in helping these receptors bind their natural ligands. These include CCR5, CXCR4, CCR2b, and CX3CR1 as well as Duffy antigen receptor for binding chemokines [14][15][16][17]29] . Previous work has indicated that sulfated tyrosines in the N-terminus of chemotactic C5aR help to form the docking site for C5a, but not the activation site [18] .…”

“…Posttranslational modification occurs in chemokine and chemotactic receptors, including C3aR, C5aR, CCR5, CX3CR1, and CC2b [15][16][17][18][19] . We hypothesized that tyrosine sulfation might be a universal phenomenon in other 7TMS GPCRs, such as ETRA and ETRB [38] .…”

“…Tyrosine sulfation tends to occur in acidic regions of molecules, usually in regions containing multiple tyrosines [12,13] . Chemokine receptors, including CCR5, CXCR4, CX3CR1 and CCR2b, are sulfated on their N-terminal tyrosines, and tyrosine sulfation is critical for ligand binding [14][15][16][17] . For example, sulfated tyrosines of the amino terminus of CCR5 contribute to the binding of CCR5 to MIP-1α, MIP-1β, and HIV-gp120/CD4 complexes; in addition, they facilitate the entry of HIV-1 into cells expressing CCR5 and CD4 [14] .…”

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

“…In a HEK293 cell line transfected to express CCR2, mutation of Tyr 26 resulted in a dramatic decrease in sulfation, a 10-fold decrease in binding affinity for MCP-1 and almost complete loss of activation in response to MCP-1 (5). Because the Tyr 26 mutant had no detectable sulfation, the authors concluded that Tyr 28 on CCR2 is not sulfated.…”

“…This modification is catalyzed in the Golgi apparatus by the enzymes tyrosylprotein sulfotransferases 1 and 2, which preferentially sulfate tyrosine residues located near acidic residues (4), a motif found in the N-terminal regions of most chemokine receptors (3). The chemokine receptors CCR2, CCR5, CCR8, CXCR3, CXCR4, CX 3 CR1, and Duffy antigen and receptor for chemokines (DARC) have been demonstrated to contain sulfated tyrosine residues that modulate chemokine binding (5)(6)(7)(8)(9)(10)(11). Moreover, studies using differentially sulfated N-terminal peptides from various chemokine receptors have shown that tyrosine sulfation increases the binding affinity of the receptor peptides to their cognate chemokines (12)(13)(14)(15)(16).…”