RESUMO
Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Receptores de GABA-B/isolamento & purificação , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Ratos , Receptores de GABA-B/genéticaRESUMO
Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/química , Animais , Ácido Aspártico Endopeptidases/química , Sítios de Ligação/fisiologia , Células COS , Clonagem Molecular , Endopeptidases , Feminino , Glicoproteínas/análise , Humanos , Masculino , Proteínas de Membrana/análise , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.