RESUMEN
Pyrokinins (PKs) are pleiotropic neuropeptides with significant roles in invertebrate physiology. Although functions of PKs are known in insects, there is a lack of knowledge of PK-encoding genes and PKs functions in ticks. Herein the first tick cDNAs of the capability (capa) gene were cloned from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae), and the blacklegged tick, Ixodes scapularis. Each cDNA encoded one periviscerokinin and five different pyrokinins. Two PKs were identical in sequence in the two species. The three PKs unique to R. microplus (Rhimi-CAPA-PK1, -PK2, and -PK5) were tested on the recombinant R. microplus pyrokinin receptor using a calcium bioluminescence assay. The Rhimi-CAPA-PKs acted as agonists with EC50s ranging from 101-188 nM. Twenty PK analogs designed for enhanced bioavailability and biostability were tested on the receptor. Five of these were designed based on the sequences of the three unique Rhimi-CAPA-PKs. Eight PK analogs were also agonists; four of them were full agonists that exhibited comparable efficacy to the native Rhimi-CAPA-PKs, with EC50 ranging from 401 nM-1.9 µM. The structure-activity relationships (SAR) of all analogs were analyzed. Our results suggested that a positively charged, basic lysine at the variable position X of the PK active core (FXPRLamide) conferred enhanced affinity to the analogs in their interaction with the tick receptor. These analogs are promising tools to elucidate the pyrokinin function in ticks in vivo as these analogs are expected to have prolonged hemolymph residence time in comparison to the native peptides.
Asunto(s)
Proteínas de Artrópodos/genética , ADN Complementario/genética , Ixodes/fisiología , Neuropéptidos/fisiología , Rhipicephalus/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Clonación Molecular , Neuropéptidos/química , Neuropéptidos/metabolismo , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/agonistas , Relación Estructura-ActividadRESUMEN
Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>>P4>T3>>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.
Asunto(s)
Proteínas de Insectos/fisiología , Lepidópteros/metabolismo , Neuropéptidos/metabolismo , Receptores de Neuropéptido/fisiología , Aequorina/genética , Secuencia de Aminoácidos , Animales , Células CHO , Señalización del Calcio/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Cricetinae , Cricetulus , ADN Complementario/química , ADN Complementario/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Hormonas de Insectos/farmacología , Proteínas de Insectos/agonistas , Proteínas de Insectos/genética , Lepidópteros/genética , Datos de Secuencia Molecular , Neuropéptidos/farmacología , Filogenia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Receptores de Neuropéptido/agonistas , Receptores de Neuropéptido/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.