Adipokinetic Hormone Receptor Research Articles

Adipokinetic hormones in the African malaria mosquito, Anopheles gambiae: Identification and expression of genes for two peptides and a putative receptor

Adipokinetic hormones (AKHs) are neuropeptides that mobilize stored fuels for flight in insects, and thus, may regulate flight by mosquitoes that transmit pathogens of human diseases. Our study of AKHs in the African malaria mosquito, Anopheles gambiae, identified and characterized the expression of genes encoding two AKHs, Anoga-AKH-I (pQLFTPAWa) and Anoga-AKH-II (pQVTFSRDWNAa), and a putative homolog for an AKH G-protein coupled receptor. Gene transcripts for both Anoga-AKHs and the AKH receptor were present in eggs, larvae, pupae, and adults of An. gambiae. In females, these transcripts were apparent in heads and thoraces for up to 72 h after blood or sugar feeding, as revealed by RT-PCR. With immunocytochemistry, a cluster of neurosecretory cells posterior to the corpus cardiacum and specific cells in the brain and thoracic ganglia of females were immunostained with an AKH antibody, which recognizes both Anoga-AKHs. Accordingly, Anoga-AKH-I was detected in extracts of female heads and thoraces by HPLC and an AKH radioimmunoassay, whereas Anoga-AKH-II was detected only in heads.

Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae

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 −9 M), a corazonin receptor (EC 50 for A. gambiae corazonin, 4 × 10 −9 M), and a crustacean cardioactive peptide (CCAP) receptor (EC 50 for A. gambiae CCAP, 1 × 10 −9 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.

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC 50, 5 × 10 −9 M), and Pea-AKH-2 (EC 50, 2 × 10 −9 M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.

Differential Receptor Activation by Cockroach Adipokinetic Hormones Produces Differential Effects on Ion Currents, Neuronal Activity, and Locomotion

Adipokinetic hormone (AKH) peptides in insects serve the endocrine control of energy supply. They also produce, however, neuronal, vegetative, and motor effects, suggesting that AKHs orchestrate adaptive behavior by multiple actions. We have cloned, for Periplaneta americana, the AKH receptor to determine its localization and, based on current measurements in neurons and heterologous expression systems, the mechanisms of AKH actions. Apart from fat body, various neurons express the AKH receptor, among them abdominal dorsal unpaired median (DUM) neurons, which release the biogenic amine octopamine. They are part of the arousal system and are involved in the control of circulation and respiration. Both the two Periplaneta AKHs activate the Gs pathway, and AKH I also potently activates Gq. AKH I and--with much less efficacy--AKH II accelerate spiking of DUM neurons through an increase of the pacemaking Ca2+ current. Because the AKHs are released from the corpora cardiaca into the hemolymph, they must penetrate the blood-brain barrier for acting on neurons. That this happens was shown electrophysiologically by applying AKH I to an intact ganglion. Systemically injected AKH I stimulates locomotion potently in striking contrast to AKH II. This behavioral difference can be traced back conclusively to the different effectiveness of the AKHs on the level of G proteins. Our findings also show that AKHs act through the same basic mechanisms on neuronal and nonneuronal cells, and they support an integration of metabolic and neuronal effects in homoeostatic mechanisms.

Molecular cloning and functional expression of a [formula omitted] corazonin receptor

The insect adipokinetic hormones (AKHs) constitute a large family of neuropeptides that mobilize lipids and sugar from the insect fat body during energy-requiring activities such as flight. We have previously identified the first insect AKH receptors from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori (Staubli et al., PNAS 2002, 99: 3446–3451). Here, we have cloned the cDNA of a Drosophila G protein-coupled receptor that was closely related to the first Drosophila AKH receptor both with respect to amino-acid sequence and gene structure. We have subsequently expressed this orphan receptor in Chinese hamster ovary cells and identified Drosophila corazonin as the endogenous ligand for the receptor. Corazonin increases heart beat in some insects, but its function in Drosophila is unknown. These results are intriguing, because not only are the Drosophila AKH and corazonin receptors structurally and evolutionarily related, but also are their preprohormones, which suggests a co-evolution of ligands and receptors. The Drosophila corazonin receptor is expressed in embryos, larvae, pupae, and adult flies. Furthermore, a receptor that is structurally very similar to the Drosophila corazonin receptor can be found in the genomic database from the malaria mosquito Anopheles gambiae.

Molecular identification of the insect adipokinetic hormone receptors.

The insect adipokinetic hormones (AKHs) are a large family of peptide hormones that are involved in the mobilization of sugar and lipids from the insect fat body during energy-requiring activities such as flight and locomotion, but that also contribute to hemolymph sugar homeostasis. Here, we have identified the first insect AKH receptors, namely those from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori. These results represent a breakthrough for insect molecular endocrinology, because it will lead to the cloning of all AKH receptors from all model insects used in AKH research, and, therefore, to a better understanding of AKH heterogeneity and actions. Interestingly, the insect AKH receptors are structurally and evolutionarily related to the gonadotropin-releasing hormone receptors from vertebrates.

Characterization of the adipokinetic hormone receptor from the fat body of Manduca sexta

A tritium labeled Manduca sexta adipokinetic hormone (M-AKH) was synthesized (pE-L-T-[p 3H]F-T-S-S-W-G-NH 2) (specific activity 27 Ci/mmol) which was fully active in a bioassay. It was used in a filtration based binding assay to characterize the M-AKH receptor from the fat body of M. sexta. Membrane fractions were prepared from fat body and optimal binding conditions were determined. A K d of 7·10 −10 M was determined and the receptor concentration estimated to be 0.5 pmol/mg membrane protein. No receptor binding was found when membranes were prepared from brain, heart or flight muscle of M. sexta or from fat body of the cockroach Blaberus discoidalis. However, specific binding was found with membrane preparations from the pterothoracic ganglion of M. sexta. The membranes from the ganglion had a much smaller number of binding sites than the fat body membranes, however, the binding was specific and observed in each experiment.

Stimulation of glycogenolysis by three locust adipokinetic hormones involves G s and cAMP

Insect adipokinetic hormones (AKHs) have been shown to mobilize fat body carcohydrate by glycogen phosphorylase activation. In this study, the signal transduction pathways of AKH-I, -II and -III from the migratory locust are further elucidated. We show that the AKHs enhance fat body cAMP levels in vitro. For all hormones, maximal levels are reached after 1 min and correspond to a 200% increase compared to resting levels. Although cAMP levels induced by massive doses of AKH-I, -II and -III are equal, AKH-III is the most potent when applied in a physiological dose. This difference in potency also applies to glycogen phosphorylase activation. Cholera toxin (CTX) likewise enhances cAMP levels and phosphorylase activity, however pertussis toxin (PTX) has no effect. Increases induced by CTX and AKH are not additive, suggesting that they share the same pathway. Phosphorylase activation by the AKHs is strongly attenuated by guanosine-5′- O-(2-thiodiphosphate) (GDPβS). These results demonstrate a role for cAMP in AKH signal transduction and indicate that the AKH receptor(s) are coupled to cAMP formation and glycogen phosphorylase activation via the stimulatory guanine nucleotide-binding protein (G s).

The pharmacology of the lipid-mobilizing response to adipokinetic hormone family peptides in the moth, Manduca sexta

The biological activities of the nonapeptide Manduca adipokinetic hormone were investigated in the tobacco hornworm. In adult moths injection of Manduca adipokinetic hormone caused an increase in haemolymph lipid concentration, while in fifth-instar larvae the peptide activated fat body glycogen phosphorylase. The dose of peptide required to cause a 50% response was 0.3 pmol (equivalent to about 0.12 ng g −1) in the adult insect, and 0.07 pmol (about 0.008 ng g −1) in a fifth-stage larva. Thus larvae were more sensitive to the hormone than adult insects. The pharmacology of the adipokinetic response in adult Manduca was investigated using naturally occurring adipokinetic hormone family peptides. Of the 11 peptides assayed only six elicited significant lipid-mobilization at a dose of 50 pmol. Of these, prawn red pigmentconcentrating hormone and Blaberus hypertrehalosaemic hormone were the least active and did not promote significant hyperlipaemia below this high dose. Locust adipokinetic hormone I, Carausius hypertrehalosaemic factor II and cockroach myoactive peptide II were all active at low doses, but were partial agonists incapable of eliciting full adipokinetic responses at doses up to 50 pmol. Other than Manduca adipokinetic hormone, only the decapeptide Heliothis hypertrehalosaemic hormone was a full agonist. This peptide was as active as Manduca adipokinetic hormone itself. The adult Manduca fat body adipokinetic hormone receptor appears to have more specific structural requirements than the Orthopteran receptors studied previously. In addition to the standard adipokinetic hormone framework of pGlu 1, Phe 4, Trp 8, and the C-terminal amide, the residues Leu 2, Thr 3, Ser 6 and Gly 9 all seem to be important for full adipokinetic activity in Manduca. Unlike the Orthopteran receptors, the Manduca receptor does not require the residue Pro 6, preferring Ser 6. A tentative model of the Manduca adipokinetic hormone receptor is proposed. A competitive adipokinetic assay showed that two adipokinetic hormone family peptides that are partial agonists ( Carausius hypertrehalosaemic factor II and locust adipokinetic hormone I) were capable of significantly reducing the lipid mobilizing response to coinjected Manduca adipokinetic hormone. This result shows that antagonists to insect peptide hormones are possible, and suggest that in adult Manduca fat body a single receptor population may be responsible for mediating the adipokinetic response.