Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae q Martin Belmont, Giuseppe Cazzamali, Michael Williamson, Frank Hauser, Cornelis J.P. Grimmelikhuijzen * Center for Functional and Comparative Insect Genomics, Department of Cell Biology and Comparative Zoology, Institute of Biology, University of Copenhagen, DK-2100 Copenhagen, Denmark Received 16 March 2006 Abstract The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four evolutionarily related G protein-coupled receptors (GPCRs) from this mosquito and expressed them in Chinese hamster ovary cells. After screening of a library of thirty-three insect or other invertebrate neuropeptides and eight biogenic amines, we could identify (de-orphanize) three of these GPCRs as: an adipokinetic hormone (AKH) receptor (EC 50 for A. gambiae AKH, 3 · 10 À9 M), a corazonin receptor (EC 50 for A. gambiae corazonin, 4 · 10 À9 M), and a crustacean cardioactive peptide (CCAP) receptor (EC 50 for A. gambiae CCAP, 1 · 10 À9 M). The fourth GPCR remained an orphan, although its close evolutionary relationship to the A. gambiae and other insect AKH receptors suggested that it is a receptor for an AKH-like peptide. This is the first published report on evolutionarily related AKH, corazonin, and CCAP receptors in mosquitoes. Ó 2006 Elsevier Inc. All rights reserved. Keywords: GPCR; DRY motif; Neuropeptide; Neurohormone; GnRH; Endocrinology; Malaria; Insect; Drosophila; Mosquito; Silkworm Malaria is one of the most serious parasitic diseases in the world, responsible for up to 500 million cases of illness and 2.7 million deaths (mostly children) each year. The dis- ease is caused by an infection with the intracellular parasite Plasmodium falciparum, which is transmitted by the malar- ia mosquito, Anopheles gambiae [1]. To find new strategies for malaria control, both the genomes from P. falciparum and that from A. gambiae were sequenced recently [2,3]. Exploiting these genomic data will open up new possibili- ties to fight P. falciparum or its vector A. gambiae with high selectivity and low human toxicity. G protein-coupled receptors (GPCRs) are well-known drug targets. More than 30% of all prescribed human med- ication is acting on this large family of membrane proteins [4]. GPCRs are structurally characterized by the presence of seven transmembrane a-helices, and they have a key function in cell communication by linking extracellular sig- nalling substances (hormones or neurotransmitters) to intracellular effector proteins. In all animals, GPCRs and their ligands steer important physiological processes such as development, reproduction, feeding, and behavior. By analysis of the sequenced Anopheles genome, 276 genes could be annotated to code for GPCRs. Twenty-five of these GPCRs were annotated as putative neuropeptide receptors belonging to the family of rhodopsin-like or class A GCPRs [5,6]. Until today, nearly all of these neuropep- tide receptors are orphans, because their ligands are 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.117 q The sequence data of this paper have been submitted to the GenBank database under Accession Nos. AY298745, AY301275, AY500851, and AY553322. * Corresponding author. Fax: +45 3532 1200. E-mail address: cgrimmelikhuijzen@bi.ku.dk (C.J.P. Grimmelikhuijzen). URL: http://www.bi.ku.dk/staff/staff-vip-details.asp?ID=90 (C.J.P. Grimmelikhuijzen). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 344 (2006) 160–165 BBRC