RESUMO
The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Receptores do Hormônio Hipofisário/química , Receptores do Hormônio Hipofisário/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/genética , Relação Estrutura-AtividadeRESUMO
Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.
Assuntos
Processamento Alternativo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , AMP Cíclico/metabolismo , Éxons , Feto/anatomia & histologia , Feto/metabolismo , Humanos , Dados de Sequência Molecular , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Isoformas de Proteínas/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Alinhamento de Sequência , Peptídeo Intestinal Vasoativo/genéticaRESUMO
In Drosophila, the three yolk protein (yp) genes are transcribed in a sex-, tissue- and developmentally specific manner, providing an ideal system in which to investigate the factors involved in their regulation. The yolk proteins are synthesized in the fat body of adult females, and in the ovarian follicle cells surrounding the developing oocyte during stages 8-10 of oogenesis. We report here an analysis of the yolk protein 3 (yp3) gene and its flanking sequences by means of P-element mediated germ-line transformation and demonstrate that a 747 bp promoter region is sufficient to direct sex-specific expression in the female fat body and both the temporal- and cell-type-specificity of expression during oogenesis. Two elements that independently govern yp3 transcription in these tissues have been separated and no other sequences in the upstream, downstream or coding regions have been identified that are autonomously involved in yp3 expression.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Proteínas do Ovo/genética , Regulação da Expressão Gênica , Genes de Insetos , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Drosophila melanogaster/genética , Proteínas do Ovo/química , Corpo Adiposo/química , Corpo Adiposo/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Ovário/química , Ovário/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Caracteres Sexuais , Transcrição GênicaRESUMO
The three yolk protein genes (yps) of Drosophila melanogaster are expressed in the ovary and fat body of the adult female. Their levels of expression in the fat body depend upon both juvenile hormone (JH) and 20-hydroxyecdysone (20E). Using transformed lines of flies with various flanking sequences from the yp genes and lacZ, Adh, or native yp genes as reporters, the regulation of the three yp genes by 20E and the JH analogue ZR515 (methoprene) was investigated. For 20E, induction of reporter gene expression in males was assayed and, for JH, upregulation of the genes in nutritionally deprived females, which express yolk proteins (YPs) at very low levels, was followed. We were able to map 20E inducible sites upstream of yp3 and sites located 3' and within the coding sequence or introns of yp3 which can interact to respond to 20E. There are also sites in the intergenic spacer between yp1 and yp2. Evidence for repressors was also found upstream of the yp genes, suggesting downstream 20E inducible elements may be important in vivo. There appears to be a difference in the response to 20E in the fat body of the thorax and abdomen between different constructs in males. It is not clear whether those sequences which respond to 20E are genuine ecdysone response elements (i.e., binding sites for the ecdysone receptor) or if the effect is indirect. Methoprene upregulation of YPs, however, was only ever observed using native yp genes as reporters, suggesting that this hormone may act on intron sequences or yp coding sequences, or perhaps by influencing stability of the yp mRNA.
Assuntos
Drosophila melanogaster/genética , Ecdisterona/farmacologia , Proteínas do Ovo/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , DNA Ribossômico/genética , Proteínas do Ovo/biossíntese , Corpo Adiposo , Feminino , Genes Reporter/genética , Masculino , Metoprene/farmacologia , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas RepressorasRESUMO
The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide are strong activators of adenylate cyclase, but recent evidence suggests that they can elicit a number of additional intracellular signals. Some of these are likely to be downstream of the conventional adenylate cyclase pathway, but it is now clear that others reflect novel primary coupling events of the receptors.