RESUMO
The mouse serotonin (5-HT) receptor subtype, 5-HT7, belongs to the family of seven transmembrane G-protein-coupled receptors. To identify the structural basis for the coupling of 5-HT7 receptor to G alpha(s) we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild-type and mutant 5-HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5-HT7(E325G) and 5-HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C-terminal region of the third intracellular loop in receptor-G-protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Serotonina/genética , Animais , Baculoviridae/genética , Linhagem Celular , Clonagem Molecular , Camundongos , Mutagênese , Conformação Proteica , Ensaio Radioligante , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , SpodopteraRESUMO
Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5-HTdro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the G(alpha)s G-protein subtype. The Drosophila 5-HTdro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin-insensitive G-protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G-proteins revealed that Sf9 cells lack the G-protein subtypes G(alpha i 1-3) and G(alpha)o, but express the subtype G(alpha)s and G(alpha)q.
Assuntos
Drosophila/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Serotonina/metabolismo , Animais , Baculoviridae , Encéfalo/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas de Drosophila , Feminino , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Immunoblotting , Inositol/metabolismo , Cinética , Glicoproteínas de Membrana/biossíntese , Miométrio/metabolismo , Gravidez , Ratos , Receptores de Serotonina/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Serotonina/metabolismo , Spodoptera , TransfecçãoRESUMO
Segments of nicotinic acetylcholine receptor alpha subunit genes have been isolated from a panel of insect species by polymerase chain reaction, using degenerate oligonucleotide primers designed to recognize conserved regions of the Drosophila melanogaster ALS and SAD genes. The amplified segments encode elements of typical alpha-subunits anticipated to play roles in ligand binding and ion channel formation. Each is also clearly either ALS or SAD-like. The predicted protein sequences display extremely high levels of conservation (over 85% for each subtype) even though derived from very distantly related insect species.