Expression cloning of an insect diuretic hormone receptor. A member of the calcitonin/secretin receptor family.

J.D Reagan

doi:10.1016/s0021-9258(17)42299-x

Abstract

Insect diuretic hormones and their receptors regulate fluid and ion secretion and thus are attractive targets for the design of novel insect control agents. A complementary DNA clone encoding a corticotropin-releasing factor-related diuretic hormone receptor from the tobacco hornworm Manduca sexta was isolated by expression cloning in COS-7 cells. The receptor consists of 395 amino acids and contains seven putative transmembrane domains. The expressed receptor binds M. sexta diuretic hormone, as well as several related insect diuretic peptides with high affinity. Furthermore, each of these peptides stimulate adenylate cyclase in COS-7 cells transfected with the receptor. The M. sexta diuretic hormone receptor is homologous to the receptors for calcitonin, secretin, vasoactive intestinal peptide, parathyroid hormone, glucagon-like peptide 1, growth hormone-releasing hormone, pituitary adenylate cyclase-activating polypeptide, and glucagon. The M. sexta diuretic hormone receptor is the first nonmammalian member of this family to be identified.

Highlights

Werefound to contain a receptor that bound3H-labeled Mas-DH with extraordinarilyhigh affinity (& = 79 PM).I have used an expression cloning strategyin COS-7 cells to isolate a cDNA that encodes a M.sexta diuretic hormone receptor
The Mas-DH receptor is the first insect-specific neuropeptide receptor to be cloned and is homologous to a recently discovered family of G-protein-coupled receptors first identified in mammals
Growth hormone-releasing hormone, glucagon, nor parath ~ i dhormone inhibited binding of 3H-labeled Mas-DH to the recombinant Mas-DH receptor at concentrations as high as 1, Tb determine if the expressed receptor could stimulate adenylate cyclase, COS-7cells transfected with pKSliMas-DH-R were incubated with Mas-DH or related peptides (Fig. 4)

Summary

Expression Cloningof an Insect Diuretic Hormone Receptor

While considerable efforthas been directed toward identifying insect diuretic hormonaesnd their modes of action, a report characterizing an insect diuretichormonereceptor has appeared only recently [8].Mt membranes from M.sexta larvae. (Received for publication, September 21, 1993, and in revised form, October 18, 1993). Werefound to contain a receptor that bound3H-labeled Mas-DH with extraordinarilyhigh affinity (& = 79 PM).I have used an expression cloning strategyin COS-7 cells to isolate a cDNA that encodes a M.sexta diuretic hormone receptor. The expressed receptorbinds Mas-DH with high affinity, stimulates. From SandozAgro, Inc., Research Division, protein-coupled receptor family

EXPERIMENTAL PROCEDURES

RESULTS

ISC CUSS ION