Functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: Evidence suggesting that the N-terminal region of diuretic hormone is associated with receptor activation

Abstract

A recombinant baculovirus containing the diuretic hormone receptor cDNA from Manduca sexta was constructed. When Spodoptera frugiperda (Sf9) cells were infected with the virus, a time-dependent expression of the receptor appeared. The expressed receptor displayed high affinity for diuretic hormone, which was similar to the affinity observed in Malpighian tubules and transfected COS-7 cells. The receptor expression level was 77 pmol/mg protein, as compared to 3.1 pmol/mg protein in Malpighian tubules and 1.3 pmol/mg protein in transfected COS-7 cells. Chemical crosslinking of 125I-labeled Mas-DH to the expressed receptor revealed a protein of 48–52 kDa. Furthermore, Mas-DH stimulated cAMP synthesis in recombinant baculovirus infected Sf9 cells. The N-terminal truncated analog [13–41] Mas-DH bound to the expressed receptor with high affinity but did not stimulate cAMP synthesis. This suggests that the N-terminal region of Mas-DH is required for receptor activation but not receptor binding. The recombinant baculovirus provides an alternate system to study receptor function and will allow large scale production of receptor for biophysical characterization.