TAILIEUCHUNG - Báo cáo khoa học: Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1–Gi2a 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–Gi2a 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. | Eur. J. Biochem. 271 1677-1689 2004 FEBS 2004 doi Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1-Gi2a fusion protein after heterologous production in baculovirus-infected insect cells Yoshitake Maeda1 2 3 Ryota Kuroki2 3 Winfried Haase4 Hartmut Michel1 and Helmut ReMander1 1 Max-Planck-Institut fur Biophysik Abt. Molekulare. Membranbiologie Frankfurt Main Germany 2Kirin Brewery Co. Ltd Pharmaceutical Division Pharmaceutical Research Laboratories Miyahara Takasaki Gunma Japan 3Kirin Brewery Co. Ltd Central Laboratories for Key Technology Kanazawa-ku Yokohama Japan 4Max-Planck-Institut fur Biophysik Abt. Strukturbiologie Frankfurt Main Germany In order to perform biochemical and pharmacological characterization of CXCR1 we designed several CXCR1 constructs. All constructs including a CXCR1-Gi2a fusion protein were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of 125I-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 Gi2 protein as a result of coinfection with baculoviruses encoding the Gi2a- the b- and the y- sub units the Bmax values were significantly increased. Hence the level of FlagCXCR1Bio after coproduction with Gi2 protein was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However no differences in the K 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 .

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