# $$Leu128^{3.43}$$ (L128) and $$Val247^{6.40}$$ (V247) of CXCR1 Are Critical Amino Acid Residues for G Protein Coupling and Receptor Activation

 Title: $$Leu128^{3.43}$$ (L128) and $$Val247^{6.40}$$ (V247) of CXCR1 Are Critical Amino Acid Residues for G Protein Coupling and Receptor Activation Author: Boisvert, William A.; Han, Xinbing; Tachado, Souvenir D.; Koziel, Henryk Note: Order does not necessarily reflect citation order of authors. Citation: Han, Xinbing, Souvenir D. Tachado, Henry Koziel, and William A. Boisvert. 2012. $$Leu128^{3.43}$$ (L128) and $$Val247^{6.40}$$ (V247) of CXCR1 are critical amino acid residues for G protein coupling and receptor activation. PLoS ONE 7(8): e42765. Full Text & Related Files: 3427349.pdf (827.9Kb; PDF) 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 $$G\alpha_{15}$$ and $$G\alpha_i$$ 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, $$Leu128^{3.43}$$ (L128) and $$Val247^{6.40}$$ (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 $$G\alpha_{15}$$. The V247N mutant also constitutively activated the $$G\alpha_i$$ 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 $$Leu128^{3.43}$$ and $$Val247^{6.40}$$ are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation. Published Version: doi:10.1371/journal.pone.0042765 Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427349/pdf/ Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:10476710 Downloads of this work: