hide
Free keywords:
CXCR1; high‐affinity binding; baculovirus expression systems
Abstract:
In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1–Gi2α 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 Gi2α‐, the β‐ and the γ‐ subunits, 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 Ki 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 Ki values remained unchanged, indicating coupling of the receptor to the guanine nucleotide‐binding protein.
