Characterization of a functional neuropeptide F receptor from Drosophila melanogaster Stephen F. Garczynski a , Mark R. Brown b , Ping Shen a , Thomas F. Murray c , Joe W. Crim a, * a Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA b Department of Entomology, University of Georgia, Athens, GA 30602, USA c Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, USA Received 30 March 2001; accepted 19 October 2001 Abstract Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125 I-NPF in a concentration- dependent manner (IC 50  65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC 50  51 nM). Whole- mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Insect; Diptera; Drosophila; G protein-coupled receptor; Neuropeptide F; Neuropeptide Y 1. Introduction Drosophila melanogaster neuropeptide F (DmNPF) is a 36 amino acid peptide found in midgut endocrine cells and the CNS of larvae and adults [4]. DmNPF is structurally related to vertebrate regulatory peptides of the neuropeptide Y (NPY) family, which includes pancreatic polypeptide (PP) and peptide YY (PYY; 4). The NPY superfamily also includes NPF’s from mollusks and platyhelminths [7,18]. In vertebrates, functions for NPY, PP, and PYY include reg- ulation of food intake, circadian rhythms, gut enzyme se- cretion and motility [5,8,9,19]. For Drosophila, in situ hy- bridization indicates DmNPF RNA occurs in midgut endocrine cells and the brain [4]. Although such positioning suggests this peptide may regulate feeding behavior and digestion, no biological functions have yet been established [4]. NPY, PYY, and PP exert their effects through a family of structurally related receptors, known as Y-receptors, that is separated into five subtypes, Y 1–2,4 –5 and y6. As for other prototypical seven transmembrane receptors, members of the NPY receptor family transduce cellular signals through interactions with G proteins. Activation of the Y-receptors by NPY-related peptides leads to an inhibition of adenylyl cyclase activity, and for the Y 1 and Y 2 receptors an increase in intracellular calcium (reviewed in 1, 2, 11). For invertebrates, two receptors resembling their NPY family counterparts have been identified and characterized [12,18]. From the snail, Lymnaea stagnalis, an NPY recep- tor homolog and its corresponding ligand, an NPF (termed “lyNPY”), have been isolated [18]. For CHO-K1 cells ex- pressing the Lymnaea receptor, addition of lyNPY inhibited adenylyl cyclase activity and elicited an increase in intra- cellular calcium, each in a dose dependent manner [18]. In Drosophila, a putative NPY receptor homolog (PR4) was cloned [12], and subsequently renamed as NepYr (see 3). For Xenopus oocytes injected with NepYr cRNA, NPY related peptides activated inward currents [12]. The poten- cies of vertebrate peptides and their analogs tested for the NepYr expressed in Xenopus oocytes were PYY  C2- NPY  NPY  [Pro 34 ]NPY  PP, a rank order atypical for any vertebrate Y-receptors [1]. Interestingly, sequence com- parison using the Drosophila receptor shows that the overall homology of NepYr to human Y 1 and Y 2 receptors is not greater than seen with unrelated mammalian G protein- coupled receptors [10]. * Corresponding author. Tel.: 1-706-542-8023; fax: 1-706-542- 4271. E-mail address: crim@cb.uga.edu (J.W. Crim). Peptides 23 (2002) 773–780 0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0196-9781(01)00647-7