RESUMO
In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Receptor 5-HT1A de Serotonina/química , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Células CHO , Linhagem Celular , Cricetinae , AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Cisteína/química , Citoplasma/metabolismo , DNA/química , Relação Dose-Resposta a Droga , Epitopos , Ésteres/química , Ácidos Graxos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hidroxilamina/farmacologia , Insetos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Células NIH 3T3 , Ácido Palmítico/química , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Fragments of two mitochondrial genes, cytochrome b (CytB) and Cytochrome c oxidase subunit I (COI) have been used as phylogenetic markers in Sergentia (Chironomidae, Diptera). The concatenated (1241 bp) sequences from both genes were used to infer the phylogenetic relationships among seven Sergentia species. Five of the species belong to the endemic fauna of Lake Baikal. Alignments of the nucleotide sequences were used for the construction of trees using Neighbor-Joining and maximum parsimony methods. Both methods yielded similar results. Monophyly of both Sergentia and the Baikalian endemic species was well supported. The date of origin of the endemic group of Sergentia was estimated as 25.7 MYA which closely coincides with the start of geological changes in the Baikal area. A cytological tree, based on 12 chromosomal characteristics, for the same set of Sergentia species showed a great similarity to the molecular phylogeny.