Leu1283.43 (L128) and Val2476.40 (V247) of CXCR1 Are Critical Amino Acid Residues for G Protein Coupling and Receptor Activation

Abstract

CXCR1, a classic GPCR that binds IL-8, plays a key role in neutrophil activation and migration by activating phospholipase C (PLC)β through Gα15 and Gαi which generates diacylglycerol and inositol phosphates (IPs). In this study, two conserved amino acid residues of CXCR1 on the transmembrane domain (TM) 3 and TM6, Leu1283.43 (L128) and Val2476.40 (V247), respectively, were selectively substituted with other amino acids to investigate the role of these conserved residues in CXCR1 activation. Although two selective mutants on Leu128, Leu128Ala (L128A) and Leu128Arg (L128R), demonstrated high binding affinity to IL-8, they were not capable of coupling to G proteins and consequently lost the functional response of the receptors. By contrast, among the four mutants at residue Val247 (TM6.40), replacing Val247 with Ala (V247A) and Asn (V247N) led to constitutive activation of mutant receptors when cotransfected with Gα15. The V247N mutant also constitutively activated the Gαi protein. These results indicate that L128 on TM3.43 is involved in G protein coupling and receptor activation but is unimportant for ligand binding. On the other hand, V247 on TM6.40 plays a critical role in maintaining the receptor in the inactive state, and the substitution of V247 impaired the receptor constraint and stabilized an active conformation. Functionally, there was an increase in chemotaxis in response to IL-8 in cells expressing V247A and V247N. Our findings indicate that Leu1283.43 and Val2476.40 are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation.

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

“…5 B and C), suggesting that the hydrophobic bridge enables helix and side chain repacking during the conformational transition. Previous mutation analyses at the 6.44 and 6.40 positions in different receptors support a role for these residues in mediating the transition between inactive and active GPCR states (3,(31)(32)(33). Whereas the identity of these residues is not absolutely conserved, the hydrophobic nature and helical compatibility of these residues is strictly conserved among all chemokine receptors (Fig.…”

The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer

“…This phenomenon is observed for virally-encoded oncogenic GPCRs 15 (Box 1) as well as many human GPCRs 16 . For example, mutations of Val247 occupying the TM6.40 position leads to constitutive activity in chemokine receptor CXCR1 17 and, quite importantly, in the thyroid stimulating hormone (TSH) receptor, TSHR, as well. In the latter receptor, mutants at Leu629 6.40 or adjacent Thr632 6.43 are among the most common TSHR mutants in thyroid cancer (Figure 2, Supplemental Tables 1-4).…”

Characterization of G protein coupling mediated by the conserved D1343.49 of DRY motif, M2416.34, and F2516.44 residues on human CXCR1

“…Our recent study highlighted the important role of amino acid residues on TM3 and TM6, especially those near intracellular loop of CXCR1, in G protein coupling and receptor activation [36]. Other Fig.…”