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Gas3/PMP22 is a tetraspan membrane protein highly expressed in myelinating Schwann cells. Point mutations in thegas3/PMP22gene account for the dominant inherited peripheral neuropathies Charcot–Marie–Tooth type 1A disease (CMT1A) and Dejerine–Sottas syndrome (DSS). Gas3/PMP22 can regulate apoptosis and cell spreading in cultured cells.Gas3/PMP22point mutations, which are responsible for these diseases, are defective in this respect. In this report, we demonstrate that Gas3/PMP22-WT is exposed at the cell surface, while its point-mutated derivatives are intracellularly retained, colocalizing mainly with the endoplasmic reticulum (ER). The putative retrieval motif present in the carboxyl terminus of Gas3/PMP22 is not sufficient for the intracellular sequestration of its point-mutated forms. On the contrary, the introduction of a retrieval signal at the carboxyl terminus of Gas3/PMP22-WT leads to its intracellular accumulation, which is accompanied by a failure to trigger cell death as well as by changes in cell spreading. In addition, by substituting the Asn at position 41 required for N-glycosylation, we provide evidence that N-glycosylation is required for the full effect on cell spreading, but it is not necessary for triggering cell death. In conclusion, we suggest that the DSS and the CMT1A neuropathies derived from point mutations ofGas3/PMP22might arise, at the molecular level, from a reduced exposure of Gas3/PMP22 at the cell surface, which is required to exert its biological functions.

Gas3/PMP22 is a tetraspan membrane protein highly expressed in myelinating Schwann cells. Point mutations in the gas3/PMP22 gene account for the dominant inherited peripheral neuropathies Charcot-Marie-Tooth type 1A disease (CMT1A) and Dejerine-Sottas syndrome (DSS). Gas3/PMP22 can regulate apoptosis and cell spreading in cultured cells. Gas3/PMP22 point mutations, which are responsible for these diseases, are defective in this respect. In this report, we demonstrate that Gas3/PMP22-WT is exposed at the cell surface, while its point-mutated derivatives are intracellularly retained, colocalizing mainly with the endoplasmic reticulum (ER). The putative retrieval motif present in the carboxyl terminus of Gas3/PMP22 is not sufficient for the intracellular sequestration of its point-mutated forms. On the contrary, the introduction of a retrieval signal at the carboxyl terminus of Gas3/PMP22-WT leads to its intracellular accumulation, which is accompanied by a failure to trigger cell death as well as by changes in cell spreading. In addition, by substituting the Asn at position 41 required for N-glycosylation, we provide evidence that N-glycosylation is required for the full effect on cell spreading, but it is not necessary for triggering cell death. In conclusion, we suggest that the DSS and the CMT1A neuropathies derived from point mutations of Gas3/PMP22 might arise, at the molecular level, from a reduced exposure of Gas3/PMP22 at the cell surface, which is required to exert its biological functions.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations ingas3/PMP22are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid–induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.