Type A Allatostatins from Drosophila melanogaster and Diplotera puncata Activate Two Drosophila Allatostatin Receptors, DAR-1 and DAR-2, Expressed in CHO Cells

• Published in: Biochemical and biophysical research communications Vol. 286; no. 5; pp. 895 - 901
• Main Authors: Larsen, Martha J, Burton, Katherine J, Zantello, Marjorie R, Smith, Valdin G, Lowery, David L, Kubiak, Teresa M
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.09.2001
• Subjects: allatostatin; Amino Acid Motifs; Animals; Ca2+ signaling; Calcium - metabolism; CHO Cells; Cloning, Molecular; Cricetinae; DAR-1; DAR-2; Diplotera punctata; Diptera; Dose-Response Relationship, Drug; Drosophila; Drosophila melanogaster; Drosophila Proteins; G-protein; GTPγS; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Insect Proteins; Kinetics; Ligands; Neuropeptides - chemistry; Neuropeptides - metabolism; Pertussis Toxin; Protein Binding; Protein Structure, Tertiary; receptor; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Signal Transduction; Time Factors; Transfection; Virulence Factors, Bordetella - pharmacology; receptor; GTPγS; Diplotera punctata; DAR-1; Ca2+ signaling; Drosophila; DAR-2; G-protein; allatostatin; CHO cells
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.5476

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH2. The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126–1131]. SRPYSFGL-NH2, (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892–5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571–577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPγS and a Ca2+ mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.