RESUMEN
Insects have two closely related G protein-coupled receptors belonging to the pyrokinin/pheromone biosynthesis activating neuropeptide (pyrokinin/PBAN) family, one with the ligand PBAN or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. A related receptor is activated by products of the capa gene, periviscerokinins. Here we characterized the PBAN receptor and the diapause hormone receptor from the European corn borer, Ostrinia nubilalis. We also identified a partial sequence for the periviscerokinin receptor. Quantitative PCR of mRNA for all three receptors indicated differential expression in various life stages and tissues. All three splice variants of the PBAN receptor were identified with all variants found in pheromone gland tissue. Immunohistochemistry of V5 tags of expressed receptors indicated that all three variants and the diapause hormone receptor were expressed at similar levels in Spodoptera frugiperda 9 (Sf9) cells. However, the A- and B-variants were not active in our functional assay, which confirms studies from other moths. Functional expression of the C-variant indicated that it is has a 44 nM half effective concentration for activation by PBAN. The diapause hormone receptor was activated by diapause hormone with a 150 nM half effective concentration.
Asunto(s)
Proteínas de Insectos/genética , Mariposas Nocturnas/genética , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Regulación de la Expresión Génica , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Especificidad de Órganos , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/metabolismo , Alineación de Secuencia , Células Sf9 , Spodoptera/genética , Spodoptera/metabolismoRESUMEN
The pyrokinin/pheromone biosynthesis-activating neuropeptide (PBAN) family of G-protein-coupled receptors and their ligands have been identified in various insects. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates, among other functions, pheromone production in moths which indicates the pleiotropic nature of these peptides. Based on the alignment of annotated genomic sequences, the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors of vertebrates. In our study, evolutionary trace (ET) analysis on the insect receptor sequences was conducted to predict the putative ligand recognition and binding sites. The ET analysis of four class-specific receptors indicated several amino acid residues that are conserved in the transmembrane domains. The receptor extracellular domains exhibit several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding.
Asunto(s)
Proteínas de Drosophila/genética , Insectos/genética , Neuropéptidos/genética , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Molecular , Insectos/metabolismo , Ligandos , Datos de Secuencia Molecular , Neuropéptidos/clasificación , Filogenia , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
The movement and dispersal of larval Lepidoptera impact their survival and distribution within the natural landscape. Homologues of the Drosophila behaviour-linked genes shaker (shkr) and slowmo (slmo) were identified from Ostrinia nubilalis (Lepidoptera: Crambidae). Onshkr was isolated as a 1610-nucleotide (nt) constitutively expressed transcript encoding a membrane-localized 469-amino-acid (aa) protein with a conserved tetramerization domain and the six-domain architecture necessary for the molecule to fold into an active K(+) channel. Three expressed splice variants of 682, 970 and 1604 nt were identified for the Onslmo gene, and encode predicted 141 and 228 aa proteins with a conserved protein of relevant evolutionary and lymphoid interest (PRELI) domain that may function in mitochondrial protein sorting and perinuclear protein localization. Onshkr and Onslmo protein sequences aligned within monophyletic lepidopteran groups.