RESUMO
By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.
Assuntos
Receptores Nicotínicos/metabolismo , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Acetilcolina/farmacologia , Alanina/química , Animais , Encéfalo/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Citoplasma/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Oócitos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Serina/química , Transfecção , Técnicas do Sistema de Duplo-Híbrido , XenopusRESUMO
High resolution structural studies of models of glutamate receptors (GluRs) have been limited to monomeric models of the ligand-binding site. To obtain oligomeric models of glutamate receptors that can reveal more complete structural information, we examined the assembly and ligand binding properties of two truncated versions of the GluR1 subunit. The first version, GluR1-WS, consisted of only the N-terminal extracellular segment (Ala(1)-Glu(520)) bridged by a synthetic linker to the second extracellular domain (Asn(615)-Gly(790)). The second version, GluR1-M1, consisted of the first N-terminal extracellular domain (Ala(1)-Glu(520)) bridged by a synthetic linker to a second segment containing the second extracellular domain, the third transmembrane domain, and the intracellular C-terminal domain (Asn(615)-Leu(889)). When expressed in Xenopus oocytes, GluR-WS was secreted and water-soluble; GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a velocity sedimentation profile that was consistent with assembly of homooligomers and bound the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high affinity. These findings show that the extracellular domains of GluR1 that are sufficient for ligand binding apparently are sufficient for subunit assembly and might be a suitable target for structural studies of a water-soluble GluR1 oligomer.
Assuntos
Receptores de AMPA/metabolismo , Animais , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de AMPA/química , Receptores de AMPA/genética , Solubilidade , Transfecção , Trítio , Água/química , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismoRESUMO
In this report, we have demonstrated that Na+/Ca2+ exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+ exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+ exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+ exchanger could make platelets especially sensitive to daily fluctuations in salt intake.