Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors.
Pheromone biosynthesis-activating neuropeptide (PBAN) is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors have been identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. No insect GPCR has been characterized with regard to role of extracellular domains required for peptide binding and receptor activation. To begin characterizing these GPCRs we created chimera receptors using a PBAN-receptor from a moth and pyrokinin-receptors from Drosophila where extracellular domains were swapped. The N-terminal of the moth GPCR has two N-glycosylation sites that when replaced with glutamines, activity was reduced but not absent, indicating these sites contribute to receptor stability. Activity was greatly reduced by replacing the 2nd extracellular loop that has an N-glycosylation site and a cysteine that can form a disulfide bridge with a cysteine at the beginning of the 3rd transmembrane domain. Exchange of the 3rd extracellular loop between the moth and Drosophila receptor resulted in differential activation by PBAN or a diapause hormone peptide. This result indicates that the 3rd extracellular loop is directly involved in peptide ligand recognition. Results are discussed in context of the structural features of insect GPCRs that are required for receptor activation as compared to vertebrate receptors.
Choi
Man-Yeon
MY
Department of Entomology, Iowa State University, 434 Science II, Ames, IA 50011-3222, USA.
Fuerst
Emily-Jean
EJ
Rafaeli
Ada
A
Jurenka
Russell
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004330:Drosophila / Q000737:chemistry* / Q000235:genetics; D005260:Female; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry* / Q000235:genetics; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics
D009479:Neuropeptides; D043562:Receptors, G-Protein-Coupled; C485184:enhanced green fluorescent protein; C079709:pheromone biosynthesis activating neuropeptide, Helicoverpa zea; C504361:pyrokinin-1 receptor, Drosophila; D049452:Green Fluorescent Proteins