RESUMO
In contrast to classical transmitters, the detailed structures and cellular and synaptic actions of neuropeptides are less well described. Peptide mass profiling of single identified neurons of the mollusc Lymnaea stagnalis indicated the presence of 17 abundant neuropeptides in the cardiorespiratory neuron, visceral dorsal 1 (VD1), and a subset of 14 peptides in its electrically coupled counterpart, right parietal dorsal 2. Altogether, based on this and previous work, we showed that the high number of peptides arises from the expression and processing of four distinct peptide precursor proteins, including a novel one. Second, we established a variety of posttranslational modifications of the generated peptides, including phosphorylation, disulphide linkage, glycosylation, hydroxylation, N-terminal pyroglutamylation, and C-terminal amidation. Specific synapses between VD1 and its muscle targets were formed, and their synaptic physiology was investigated. Whole-cell voltage-clamp analysis of dissociated heart muscle cells revealed, as tested for a selection of representative family members and their modifications, that the peptides of VD1 exhibit convergent activation of a high-voltage-activated Ca current. Moreover, the differentially glycosylated and hydroxylated alpha2 peptides were more potent than the unmodified alpha2 peptide in enhancing these currents. Together, this study is the first to demonstrate that single neurons exhibit such a complex pattern of peptide gene expression, precursor processing, and differential peptide modifications along with a remarkable degree of convergence of neuromodulatory actions. This study thus underscores the importance of a detailed mass spectrometric analysis of neuronal peptide content and peptide modifications related to neuromodulatory function.
Assuntos
Lymnaea/química , Neurônios/química , Neuropeptídeos/análise , Proteômica , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas/fisiologia , Cromatografia Líquida de Alta Pressão , Técnicas de Cocultura , Gânglios dos Invertebrados/citologia , Expressão Gênica , Glicosilação , Hidroxilação , Transporte de Íons/efeitos dos fármacos , Lymnaea/citologia , Dados de Sequência Molecular , Peso Molecular , Miócitos Cardíacos/fisiologia , Neurônios/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neuropeptídeos/fisiologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/análise , Fosfoproteínas/metabolismo , Fosfoproteínas/farmacologia , Fosforilação , Precursores de Proteínas/análise , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Synthesis of bioactive peptides is regulated by several post-translational processing events, including cleavage of peptides from a prohormone, and chemical modifications. Using quantitative in situ hybridization and neuron-specific macro-arrays, we first demonstrated cell-type specific expression levels of transcripts encoding prohormone convertases, peptide alpha-amidating enzyme as well as the chaperone 7B2 in Lymnaea neurons. Second, we demonstrated a strict correlation between alpha-amidating enzyme and its neuropeptide substrates. However, this strict relationship of gene expression of the three prohormone convertases and types of cleavage site used is not present. Third, we showed by a physiological stimulus, i.e. clean water, which leads to a stereotyped egg-laying behaviour resulting in successful egg-mass deposition, the co-regulated induction of transcript levels of processing enzymes, 7B2, and egg-laying hormone. These data indicate that (i) these enzymes (and chaperone) are involved in egg-laying hormone neuropeptide biosynthesis, and (ii) neuropeptide release and transcript levels of both prohormones and processing enzymes are regulated in accordance with physiological demands.
Assuntos
Regulação da Expressão Gênica/fisiologia , Lymnaea/metabolismo , Oxigenases de Função Mista/genética , Chaperonas Moleculares/genética , Complexos Multienzimáticos/genética , Neurônios/metabolismo , Neuropeptídeos/genética , Pró-Proteína Convertases/genética , Animais , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Oxigenases de Função Mista/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/metabolismo , Neurônios/citologia , Neuropeptídeos/metabolismo , Estimulação Física , Pró-Proteína Convertases/metabolismo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi-endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (psi-psi-X-X-phi) defining minimum requirements for GGA binding to cytoplasmic receptor domains.
