Abstract
The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.
Highlights
Binding of [fluorescein-Trp25]glucagon to Schneider 2 (S2) cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity
The high levels of expression of human glucagon receptor obtained in S2 cells have allowed us to directly monitor changes in the fluorescence properties of this ligand as it binds to the receptor
Expression of the Human Glucagon Receptor in S2 Cells— The human glucagon receptor cDNA was subcloned into vector pRmHa3 and cotransfected with pUChsneo into S2 cells. pRmHa3 contains a Drosophila metallothionein promoter that is tightly regulated in S2 cells [10]
Summary
Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. The high levels of expression of human glucagon receptor obtained in S2 cells have allowed us to directly monitor changes in the fluorescence properties of this ligand as it binds to the receptor. The glucagon receptor shares significant sequence homology with the subfamily of G protein-coupled receptors that includes receptors for glucagonlike peptide-1 and parathyroid hormone (for review, see Ref. 6) This subclass of G protein-coupled receptors bears little sequence homology to the well characterized -adrenergic/rhodopsin subfamily, and relatively little is known about the molecular interactions of ligands with receptors in this class. The concentration of antibody was adjusted so that 10 l quenched 90% of the fluorescence of 1 ml of a 5 nM fluorescein solution at pH 8.0
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