Cysteine residues are critical for chemokine receptor CXCR2 functional properties.
We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.
Limatola
Cristina
C
Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza BEMM, Università La Sapienza, I-00185 Roma, Italy. cristina.limatola@uniroma1.it
Di Bartolomeo
Sabrina
S
Catalano
Myriam
M
Trettel
Flavia
F
Fucile
Sergio
S
Castellani
Loriana
L
Eusebi
Fabrizio
F
D019943:Amino Acid Substitution; D001667:Binding, Competitive; D019921:Biotinylation; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D004220:Disulfides; D019707:Receptors, Chemokine; D003545:Cysteine; D002118:Calcium