RESUMO
Embryonic sensory neurons express membrane-anchored growth factors that stimulate proliferation and differentiation of Schwann cells. The most important of these are members of the neuregulin-1 (Nrg-1) family that activate the erbB2/erbB3 receptor kinase on Schwann cells. Nrg-1 growth factors display a complex pattern of alternative mRNA splicing. We investigated the expression of the Nrg-1 type I in rat embryo dorsal root ganglion (DRG) neurons. Nrg-1 type I mRNA was abundantly expressed in DRG neurons; molecular cloning identified three distinct isoforms. The most prominent structural difference produced by alternative splicing was truncation of the C-terminal cytoplasmic domain. In sensory neurons and other cells, Nrg-1 type I proteins with the full-length 374-amino-acid cytoplasmic domain were expressed on the cell surface. In contrast, an isoform with a partially truncated cytoplasmic domain was retained in an intracellular compartment. Deletion studies demonstrated the presence of a cryptic intracellular retention signal that was exposed in the truncated cytoplasmic domain. Cell surface Nrg-1 type I molecules were subject to protease-dependent release of the biologically active ectodomain. As a consequence of their intracellular localization, the Nrg-1 type I isoform with a truncated cytoplasmic domain was not subject to membrane shedding. Nrg-1 type I ectodomain release was accelerated by factors present in Schwann cell-conditioned medium. In cells with active Nrg-1 type I ectodomain, shedding products corresponding to the cytoplasmic domain were not detected, because of rapid gamma-secretase- and proteasome-dependent degradation. These results demonstrate that sensory neurons express alternatively spliced neuregulin polypeptides with distinct subcellular localizations and processing.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Gânglios Espinais/citologia , Neuregulina-1/metabolismo , Neurônios/citologia , Transcrição Gênica , Animais , Animais Recém-Nascidos , Brefeldina A/farmacologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Embrião de Mamíferos , Humanos , Imunoprecipitação/métodos , Neuregulina-1/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tiofenos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transfecção/métodosRESUMO
Schwann cell myelination requires interactions with the extracellular matrix (ECM) mediated by cell surface receptors. Previously, we identified a type V collagen family member, alpha4(V) collagen, which is expressed by Schwann cells during peripheral nerve differentiation. This collagen binds with high affinity to heparan sulfate through a unique binding motif in the noncollagenous N-terminal domain (NTD). The principal alpha4(V) collagen-binding protein on the Schwann cell surface is the heparan sulfate proteoglycan glypican-1. We investigated the role of alpha4(V) collagen and glypican-1 in Schwann cell terminal differentiation in cultures of Schwann cells and dorsal root ganglion neurons. Small interfering RNA-mediated suppression of glypican-1 expression decreased binding of alpha4(V)-NTD to Schwann cells, adhesion and spreading of Schwann cells on alpha4(V)-NTD, and incorporation of alpha4(V) collagen into Schwann cell ECM. In cocultures, alpha4(V) collagen coassembles with laminin on the surface of polarized Schwann cells to form tube-like ECM structures that are sites of myelination. Suppression of glypican-1 or alpha4(V) collagen expression significantly inhibited myelination. These results demonstrate an important role for these proteins in peripheral nerve terminal differentiation.
