Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1-Galpha fusion protein after heterologous production in baculovirus-infected insect cells.
In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.
Maeda
Yoshitake
Y
Max-Planck-Institut für Biophysik, Abt. Molekulare. Membranbiologie, Frankfurt/Main, Germany. ymaeda@kirin.co.jp
Kuroki
Ryota
R
Haase
Winfried
W
Michel
Hartmut
H
Reiländer
Helmut
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006031:Glycosylation; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D023062:Receptors, Interleukin-8A / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D018411:Spodoptera
D011518:Proto-Oncogene Proteins; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D006165:Guanylyl Imidodiphosphate; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go