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.
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.
Reagan
J D
JD
Sandoz Agro Inc., Research Division, Palo Alto, CA 94304, USA.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D017344:Genes, Insect; D007301:Insect Hormones / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide / Q000235:genetics* / Q000378:metabolism; D015290:Second Messenger Systems; D018411:Spodoptera
D018076:DNA, Complementary; D007301:Insect Hormones; D019476:Insect Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D000242:Cyclic AMP