RESUMO
G-protein-coupled receptors (GPCRs) represent a diverse protein family of receptors that transduce signals from the extracellular surrounding to intracellular signaling molecules evoking various cellular responses. It is now widely accepted that GPCRs are expressed and function as dimers or most probably as oligomers of more than two receptor protomers. The heteromer has different biochemical and pharmacological characteristics from the monomers, which increases the functional responses of GPCRs. GPCRs are involved in many diseases, and are also the target of around half of all modern medicinal drugs. In the case of Parkinson's disease, a degenerative process caused by gradual disappearance of dopaminergic nigrostriatal neurons, it is suspected that the targets for treatment should be dopamine-receptor-containing heteromers. Technologies based on the use of fluorescent- or luminescent-fused receptors and adaptations of resonance energy transfer (RET) techniques have been useful in investigating the functional inter-relationships between receptors in a heteromer. In this study functional recombinant adenosine A(2A)-Rluc, dopamine D(2)-GFP(2) and histamine H(3)-YFP receptor fusion proteins were successfully cloned and characterized, producing the essential basis for heteromerization studies between these receptors. This might provide a better insight into their pharmacological and functional inter-relationships in the brain and enable the design and evaluation of new therapeutic strategies for Parkinson's disease.
Assuntos
Multimerização Proteica/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes de Fusão/síntese química , Animais , Proteínas de Bactérias/genética , Células CHO , Cricetinae , Cricetulus , Agonistas de Dopamina/farmacologia , Desenho de Fármacos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/isolamento & purificação , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/isolamento & purificação , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos H3/química , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/isolamento & purificação , Proteínas Recombinantes de Fusão/genéticaRESUMO
Neuronal histamine regulates several functions in the vertebrate brain. The zebrafish brain contains a widespread histaminergic system and H(3) receptor ligand binding has been reported. In this study we provide evidence for the existence of histamine H(1), H(2) and H(3) receptor genes in zebrafish. Single copies of putative histamine H(1), H(2) and H(3) receptors were identified and cloned from the zebrafish brain. Expression analysis suggested that they are expressed in the brain and a few other tissues. Widespread distribution of zebrafish H(2) receptor binding sites was detected with [(125)I]iodoaminopotentidine in brain sections. Zebrafish larvae were exposed to 1, 10 or 100 microM of the H(1) ligand pyrilamine, the H(2) ligand cimetidine and the H(3) ligands thioperamide and immepip for 5 days. Significant decreases in swimming distance were observed with the highest dose of all ligands, whereas cimetidine gave a significant decrease also with 1 and 10 microM doses. These results provide the first molecular biological evidence for the presence of histamine receptors in zebrafish. These histamine receptors resemble those of higher vertebrates and they provide a useful model for pharmacological and behavioral studies for characterizing the functions of histamine in more detail.
Assuntos
Comportamento Animal/efeitos dos fármacos , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos H1/isolamento & purificação , Receptores Histamínicos H2/isolamento & purificação , Receptores Histamínicos H3/isolamento & purificação , Animais , Comportamento Animal/fisiologia , Cimetidina/farmacologia , Imidazóis/farmacologia , Piperidinas/farmacologia , Peixe-ZebraRESUMO
We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.
