RESUMO
Due to their structural characteristics and their diversity, the 22 members of the protocadherin-gamma (Pcdhgamma) family have been suggested to contribute to the establishment of specific connections in the nervous system. Here, we focus on a single isoform, Pcdhgamma-b1. Its expression is found in different brain regions and in developing spinal cord it is restricted to scattered cells, whereas all cells are labeled using an antibody that recognizes all Pcdhgamma isoforms. As a first step to understanding the signaling mechanisms downstream of Pcdhgamma, we identify the microtubule-destabilizing protein SCG10 as a cytoplasmic interactor for Pcdhgamma-b1 and other isoforms of the Pcdhgamma-b subfamily, and show that SCG10 and Pcdhgamma-b1 are found together in certain neuronal growth cones.
Assuntos
Caderinas/metabolismo , Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células COS , Proteínas Relacionadas a Caderinas , Caderinas/genética , Proteínas de Ligação ao Cálcio , Chlorocebus aethiops , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Neurônios/citologia , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Estatmina , Técnicas do Sistema de Duplo-HíbridoRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive loss of the spinal motoneurons. The SMA-determining gene has been termed survival motor neuron (SMN) and is deleted or mutated in over 98% of patients. The encoded gene product is a protein expressed as different isoforms. In particular, we showed that the rat SMN cDNA produces two isoforms with M(r) of 32 and 35kDa, both localized in nuclear coiled bodies, but the 32kDa form is also cytoplasmic, whereas the 35kDa form is also microsomal. To determine the molecular relationship between these two isoforms and potential post-translational modifications, we performed transfection experiments with a double-tagged rat SMN. Immunoblot and immunostaining studies demonstrated that the 32kDa SMN isoform derives from the full length 35kDa, through a proteolytic cleavage at the C-terminal. Furthermore, the 35kDa SMN isoform is physiologically phosphorylated in vivo. This may modulate its interaction with molecular partners, either proteins or nucleic acids.
Assuntos
Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Células 3T3 , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Humanos , Camundongos , Peso Molecular , Neurônios Motores/citologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/genética , Fosforilação , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Complexo SMN , Medula Espinal/citologia , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
Protocadherins gamma (Pcdhgamma) are a family of transmembrane proteins in which variable extracellular domains are associated with an invariant cytoplasmic domain, potentially allowing these proteins to trigger common cellular responses through diverse extracellular signals. We studied the expression of the family by in situ hybridisation and immunohistochemistry for the conserved portion of the mRNA or protein. During mouse development, Pcdhgamma expression is highest in neural tissues, but is also present in some nonneural tissues. In the adult, Pcdhgamma expression is maintained at high levels in brain, in particular in hippocampus and in the Purkinje cells of the cerebellum, whereas it is downregulated in spinal cord. Using antibodies against the conserved cytoplasmic domain, we show that in cultured embryonic spinal cord neurons, Pcdhgamma protein is present initially in both axonal and dendritic growth cones. At later stages of differentiation in vitro, Pcdhgamma distribution becomes polarised to the somatodendritic compartment. We propose that members of the Pcdhgamma family may play roles in neuronal growth and maturation.
