Singh, Satya P.; Foley, John F.; Zhang, Hongwei H.; Hurt, Darrell E.; Richards, Jennifer L.; Smith, Craig S.; Liao, Fang; Farber, Joshua M.
Selectivity in the Use of G(i/o) Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the (DRY3.51)-R-3.49-Y-3.50 sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains (DRFIV3.53)-R-3.49-F-3.50-I-3.51-V-3.52 at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individualG(i/o) proteins; and the effects of mutations in H3C on receptor function and selectivity in G(i/o) protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available G(i/o) proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments.


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