Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue

Mouse Ccr1l1 (Ccr1-like 1) encodes an orphan G-protein-coupled receptor (GPCR) with the highest homology to the inflammatory and highly promiscuous chemokine receptors Ccr1 and Ccr3 (70 and 50% amino acid identity, respectively). Ccr1l1 was first cloned in 1995, yet current knowledge of this putative chemokine receptor is limited to its gene organization and chromosomal localization. Here we report that Ccr1l1 is a Rodentia-specific gene selectively expressed in eosinophils. However, eosinophil phenotypes, development, and responsiveness to chemokines were all normal in naïve Ccr1l1 knockout mice. We demonstrate for the first time that recombinant Ccr1l1 is expressed on the plasma membrane of transfected cells and contains an extracellular N terminus and an intracellular C terminus, consistent with GPCR topology. Using receptor internalization, β-arrestin recruitment, calcium flux, and chemotaxis assays, we excluded all 37 available mouse chemokines, including Ccr1 ligands, and two viral chemokines as Ccr1l1 ligands, and demonstrated that mouse Ccr1, but not Ccr1l1, exhibits constitutive signaling activity. However, sequence analysis and structural modeling revealed that Ccr1l1 is well equipped to act as a classical signaling GPCR, with N-terminal sulfotyrosines as the only signaling and chemokine-binding determinant absent in Ccr1l1. Hereof, we show that a sulfatable N-terminal Ccr1 Y¹⁸ residue is essential for chemotaxis and calcium responses induced by Ccl3 and Ccl9/10, but substituting the corresponding Ccr1l1 F¹⁹ residue with tyrosine failed to confer responsiveness to Ccr1 ligands. Although Ccr1l1 remains an extreme outlier in the chemokine receptor family, our study supports that it might respond to unidentified mouse chemokine ligands in eosinophil-driven immune responses.