Role of Merlin/NF2 inactivation in tumor biology.

Merlin (Moesin-ezrin-radixin-like protein, also known as schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Loss of function mutations or deletions in NF2 cause neurofibromatosis type 2 (NF2), a multiple tumor forming disease of the nervous system. NF2 is characterized by the development of bilateral vestibular schwannomas. Patients with NF2 can also develop schwannomas on other cranial and peripheral nerves, as well as meningiomas and ependymomas. The only potential treatment is surgery/radiosurgery, which often results in loss of function of the involved nerve. There is an urgent need for chemotherapies that slow or eliminate tumors and prevent their formation in NF2 patients. Interestingly NF2 mutations and merlin inactivation also occur in spontaneous schwannomas and meningiomas, as well as other types of cancer including mesothelioma, glioma multiforme, breast, colorectal, skin, clear cell renal cell carcinoma, hepatic and prostate cancer. Except for malignant mesotheliomas, the role of NF2 mutation or inactivation has not received much attention in cancer, and NF2 might be relevant for prognosis and future chemotherapeutic approaches. This review discusses the influence of merlin loss of function in NF2-related tumors and common human cancers. We also discuss the NF2 gene status and merlin signaling pathways affected in the different tumor types and the molecular mechanisms that lead to tumorigenesis, progression and pharmacological resistance.

Association of beta 1 integrin with focal adhesion kinase and paxillin in differentiating Schwann cells.

Schwann cells (SCs) differentiate into a myelinating cell when simultaneously adhering to an axon destined for myelination and basal lamina. We are interested in defining the signaling pathway activated by basal lamina. Using SC/sensory neuron (N) cocultures, we identified beta1 integrin and F-actin as components of a pathway leading to myelin gene expression and myelination (Fernandez-Valle et al., 1994, 1997). Here, we show that focal adhesion kinase (FAK) and paxillin are constitutively expressed by SCs contacting axons in the absence of basal lamina. Tyrosine phosphorylation of FAK and paxillin increases as SCs form basal lamina and differentiate. FAK and paxillin specifically coimmunoprecipitate with beta1 integrin in differentiating SC/N cocultures but not SC-only cultures. Paxillin coimmunoprecipitates with FAK and fyn kinase in differentiating SC/N cocultures. A subset of tyrosine-phosphorylated beta1 integrin, FAK, and paxillin molecules reside in the insoluble, F-actin-rich fraction of differentiating cocultures. Cytochalasin D, an actin depolymerizing agent, decreases tyrosine phosphorylation of FAK and paxillin and their association with beta1 integrin and causes a dose-dependent increase in the abundance of insoluble FAK and paxillin complexes. Collectively, our work indicates that beta1 integrin, FAK, paxillin, and fyn kinase form an actin-associated complex in SCs adhering to basal lamina in the presence of axons. This complex may be important for initiating the process of SC differentiation into a myelinating cell.

Localization of focal adhesion kinase in differentiating Schwann cell/neuron cultures.

Previous studies have shown that Schwann cells (SCs) differentiate into myelin-forming or ensheathing cells only under conditions which allow the deposition of basal lamina and extracellular collagen [Bunge (1993) Peripheral Neuropathy, pp. 299-316]. SC adhesion to basal lamina is mediated by beta1 integrins and function blocking antibodies to beta1 integrins inhibit myelination [Fernandez-Valle et al. (1993) Development 119:867-880]. Recently, focal adhesion kinase (FAK), a cytoplasmic non-receptor tyrosine kinase, was found to mediate beta1 integrin-dependent signalling in a variety of cultured cell types adhering to ECM components such as fibronectin [reviewed in Schwartz et al. (1995) Ann. Rev. Cell Biol. 11:549-599; Ilic et al. (1997) J. Cell Sci. 110:401-407]. In the present study, we have determined more precisely the respective time courses of ECM deposition and myelination. In addition, we have studied by immunocytochemistry, immuno-gold labelling, and electron microscopy the expression and subcellular localization of FAK in nondifferentiating SCs and in SCs differentiating into myelinating cells. We show that the development of basal lamina and extracellular collagen fibrils precedes by 3 days the appearance of the first myelin sheaths. FAK was detected by immunocytochemistry or immuno-gold labelling only in SCs differentiating in the presence of ascorbic acid. Localization of FAK to the abaxonal plasma membrane was dependent upon ECM deposition. Cytochalasin D did not prevent or disrupt localization of FAK to the plasma membrane. These data support the possibility that FAK acts as an intermediate in the pathway by which basal lamina regulates SC differentiation.

TGF-beta1 expression is reduced in hydrocephalic H-Tx rat brain.

Transforming growth factor-beta1 (TGF-beta1) is a cytokine with diverse biological effects. Overexpression of TGF-beta1 in mice has been shown to induce progressive hydrocephalus. We have used a quantitative RT-PCR method to analyze the TGF-beta1 expression in the brains of H-Tx rat, a model of congenital hydrocephalus. Our studies have shown that rather than increased expression, the 3- and 10-day hydrocephalic H-Tx rats have significantly lower TGF-beta1 levels than their normal siblings (p < 0.01). This difference became insignificant in the 21-day group. Besides, both hydrocephalic and normal H-Tx rats have significantly lower TGF-beta1 levels in all three age groups of 3-, 10- and 21-days than SD control rats (p < 0.01 in all three groups) although the difference tends to become less significant with development. We also tested the expression of another cytokine, the epidermal growth factor, and observed a similar reduction. This suggests that the TGF-beta1 expression change is not unique to the development of hydrocephalus in this rat model. Our hypothesis is that the TGF-beta1 expression decrease in the H-Tx rat is not the cause of the disease. Rather it might be the result of feedback inhibition by increase in the expression of the gene it regulates, including an extracellular matrix component. Effort is currently being made to test this hypothesis.

LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption.

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5ß1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.

Neuregulin and laminin stimulate phosphorylation of the NF2 tumor suppressor in Schwann cells by distinct protein kinase A and p21-activated kinase-dependent pathways.

Mutations in the neurofibromatosis type 2 (NF2) gene cause formation of schwannomas and other tumors in the nervous system. The NF2 protein, Schwannomin/Merlin, is a cytoskeleton-associated tumor suppressor regulated by phosphorylation at serine 518 (S518). Unphosphorylated Schwannomin restricts cell proliferation in part by inhibiting Rac- and p21-activated kinase (Pak). In a negative-feedback loop, Pak phosphorylates Schwannomin inactivating its ability to inhibit Pak. Little is known about receptor mechanisms that promote Pak activity and Schwannomin phosphorylation. Here we demonstrate in primary Schwann cells (SCs) that Schwannomin is rapidly phosphorylated on S518 by Pak following laminin-1 binding to beta1 integrin, and by protein kinase A following neuregulin-1beta (NRG1beta) binding to ErbB2/ErbB3 receptors. These receptors, together with phosphorylated Schwannomin, P-Pak, Cdc42 and paxillin are enriched at the distal tips of SC processes, and can be isolated as a complex using beta1 integrin antibody. Dual stimulation with laminin-1 and NRG1beta does not synergistically increase Schwannomin phosphorylation because ErbB2 kinase partially antagonizes integrin-dependent activation of Pak. These results identify two parallel, but interactive pathways that inactivate the tumor suppressor activity of Schwannomin to allow proliferation of subconfluent SCs. Moreover, they identify ErbB2, ErbB3 and beta1 integrins as potential therapeutic targets for NF2.

Regulation of acetylcholinesterase synthesis and assembly by muscle activity. Effects of tetrodotoxin.

The abundance and distribution of acetylcholinesterase (AChE) oligomeric forms expressed in skeletal muscle is strongly dependent upon the activity state of the cells. In this study, we examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction of tissue-cultured quail myotubes with tetrodotoxin (TTX) reduces AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease is due primarily to the loss of 20 S asymmetric (collagen-tailed) AChE from TTX-treated cultures and is reflected in reduced pool sizes for both cell surface and intracellular AChE molecules. Using monoclonal anti-AChE antibodies to immunoprecipitate and quantify isotopically labeled enzyme molecules, we show that AChE down-regulation by TTX is not mediated through changes in the rates of synthesis or degradation of AChE polypeptide chains. Newly synthesized AChE polypeptides acquire enzymatic activity at the same rate in TTX-treated cultures as in actively contracting cells, however, a larger percentage of catalytically active dimers and tetramers are secreted from TTX-treated cultures compared with controls. These results suggest that TTX-induced down-regulation of asymmetric AChE occurs at the level of assembly of globular AChE molecules with collagen-like tail subunits in the Golgi apparatus, rather than through changes in the availability of catalytic subunits. Thus, post-translational mechanisms appear to play an important role in regulating the abundance and distribution of this important synaptic component in skeletal muscle.

Schwann cells degrade myelin and proliferate in the absence of macrophages: evidence from in vitro studies of Wallerian degeneration.

Interruption of axonal continuity in peripheral nerve trunks leads to axonal and myelin breakdown and removal distal to the injury site, a process known as Wallerian degeneration. Clearance of axonal and myelin debris has been attributed to the cooperative actions of two cell types, the indigenous Schwann cells and macrophages recruited to the regions of tissue damage. Recent work in this area has suggested a limited role for Schwann cells in myelin degradation and has emphasized the role of macrophages, not only in myelin clearance but also in the stimulation of Schwann cell proliferation which also occurs during Wallerian degeneration. In this report, we demonstrate that rat Schwann cells are capable of substantial myelin degradation unaided by macrophages. Observations were made following excision of neuronal somata from well-myelinated rat dorsal root ganglion neuron/Schwann cell co-cultures. The various stages of myelin breakdown were observed by phase microscopy, Sudan black staining, or electron microscopy. The time course for breakdown of individual myelin internodes varied from 2 to 10 days after injury and was to some extent dependent upon the original internodal length. Additionally, we show that most Schwann cells involved in Wallerian degeneration in the absence of macrophages undergo cell division following degradation of myelin into granules visible by light microscopy. The co-cultures employed were essentially free of macrophages as assessed by immunostaining for the OX42, ED2, and ED1 macrophage markers. No macrophages were detected by light or electron microscopy in the vicinity of the identified Schwann cells and furthermore, macrophages/monocytes were rarely observed in uninjured co-cultures as assessed by fluorochrome-conjugated acetylated LDL labelling. These results provide evidence in support of the ability of Schwann cells to carry out degradation of short myelin segments and to proliferate without macrophage assistance during Wallerian degeneration in vitro.

Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells.

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especially schwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlin functions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction. Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of rat sensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation into myelinating cells. Western blot analysis revealed that neurons predominantly expressed a 68-kD protein, but SCs expressed two additional 88- and 120-kD related proteins. Extensive immunological characterization demonstrated that the 88-kD protein shared three domains with the 68-kD merlin protein. Western blot analysis of soluble and insoluble culture fractions demonstrated that the majority of merlin and related proteins were soluble in isolated SCs and undifferentiated SC/N cultures, but became insoluble in myelinating SC/N cultures. Double immunofluorescence staining suggested that merlin translocated from the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalized with beta1 integrin. Finally, beta1 integrin antibody coimmunoprecipitated 68-kD merlin from isolated SC and undifferentiated SC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provide evidence that merlin interacts with beta1 integrin and that merlin localization changes from a cytosolic to cytoskeletal compartment during SC differentiation.

Allelic variants of acetylcholinesterase: genetic evidence that all acetylcholinesterase forms in avian nerves and muscles are encoded by a single gene.

Two acetylcholinesterase (AcChoEase) polypeptide chains, alpha and beta, are expressed in avian nerves and muscles with apparent molecular masses of 110 and 100 kDa, respectively. We now show that individual quails express alpha, beta, or both AcChoEase polypeptide chains. By mating studies we show that the two AcChoEase polypeptides are autosomal and segregate as codominant alleles in classical Mendelian fashion. Biochemical studies of the two allelic AcChoEase polypeptides indicate that they have the same turnover number, have the same Km for acetylcholine, are immunoprecipitated to the same extent with a monoclonal anti-AcChoEase antibody, and can assemble with equal efficiency into multimeric forms. Thus there are no obvious functional differences between the two alleles. In heterozygotes, the rates of synthesis of the two polypeptides are identical, suggesting that there are no differences in expression of these two genes. Within an individual, nerves and muscles always express the same AcChoEase forms isolated from muscle indicates that all AcChoEase forms are comprised of the same allelic polypeptide chains. In contrast to the nicotinic acetylcholine receptors that appear to be encoded by complex multigene families, our studies on AcChoEase show that all forms of this important synaptic component in electrically excitable cells are encoded by a single gene. Thus differences in assembly and localization of the multiple synaptic forms of AcChoEase must arise through posttranscriptional events, posttranslational modifications of a similar AcChoEase polypeptide chain or both.

Expression of the protein zero myelin gene in axon-related Schwann cells is linked to basal lamina formation.

A Schwann cell has the potential to differentiate into either a myelinating or ensheathing cell depending upon signals received from the axon that it contacts. Studies focusing on the pathway leading to myelination demonstrated that Schwann cells must form a basal lamina in order to myelinate an axon. In this report, we describe studies that indicate that initiation of basal lamina synthesis is required for Schwann cells to distinguish between myelination-inducing axons and axons that do not induce myelination, and to respond by undergoing the appropriate genetic and cellular changes. We have used high resolution in situ hybridization, immunocytochemistry and electron microscopy to examine changes in gene expression and morphology of Schwann cells differentiating into myelin-forming cells in vitro. These experiments were carried out in dorsal root ganglion neuron/Schwann cell co-cultures maintained in either serum-free, serum-only or serum-plus-ascorbate-containing medium. We have made four novel observations that contribute significantly to our understanding of how basal lamina and myelination are linked. (1) The addition of ascorbate (in the presence of serum), which promotes basal lamina production, appears to induce expression of the protein zero gene encoding the major structural protein of myelin. Moreover, expression of protein zero mRNA and protein, and its insertion into myelin membranes, occurs only in the subset of Schwann cells contacting myelination-inducing axons. Schwann cells in contact with axons that do not induce myelination, or Schwann cells that have not established a unitary relationship with an axon, do not express protein zero mRNA although they produce basal lamina components. (2) In serum-free conditions, a majority of Schwann cells express protein zero mRNA and protein, but this change in gene expression is not associated with basal lamina formation or with elongation of the Schwann cell along the axon and elaboration of myelin. (3) In the presence of serum (and the absence of ascorbate), Schwann cells again fail to form basal lamina or elongate but no longer express protein zero mRNA or protein. (4) Myelin-associated glycoprotein and galactocerebroside, two additional myelin-specific components, can be expressed by Schwann cells under any of the three culture conditions. Therefore, we have demonstrated that axonal induction of protein zero gene expression in Schwann cells is subject to regulation by both serum- and ascorbate-dependent pathways and that not all myelin-specific proteins are regulated in the same manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin plays a role in both changes in cell shape and gene-expression associated with Schwann cell myelination.

Schwann cell (SC) differentiation into a myelinating cell requires concurrent interactions with basal lamina and an axon destined for myelination. As SCs differentiate, they undergo progressive morphological changes and initiate myelin-specific gene expression. We find that disrupting actin polymerization with cytochalasin D (CD) inhibits myelination of SC/neuron co-cultures. Basal lamina is present, neurons are healthy, and the inhibition is reversible. Electron microscopic analysis reveals that actin plays a role at two stages of SC differentiation. At 0.75-1.0 microg/ml CD, SCs do not differentiate and appear as "rounded" cells in contact with axons. This morphology is consistent with disruption of actin filaments and cell shape changes. However, at 0.25 microg/ml CD, SCs partially differentiate; they elongate and segregate axons but generally fail to form one-to-one relationships and spiral around the axon. In situ hybridizations reveal that SCs in CD-treated cultures do not express mRNAs encoding the myelin-specific proteins 2',3'-cyclic nucleotide phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), and P0. Our results suggest that at the lower CD dose, SCs commence differentiation as evidenced by changes in cell shape but are unable to elaborate myelin lamellae because of a lack of myelin-specific mRNAs. We propose that F-actin influences myelin-specific gene expression in SCs.

Regrowth of axons in lesioned adult rat spinal cord: promotion by implants of cultured Schwann cells.

Highly purified populations of Schwann cells were grafted into lesioned adult rat spinal cord to determine if they promote axonal regeneration. Dorsal spinal cord lesions were created by a photochemical lesioning technique. Schwann cells derived from E16 rat dorsal root ganglia, either elongated and associated with their extracellular matrix or dissociated and without matrix, were rolled in polymerized collagen to form an implant 4-6 mm long which was grafted at 5 or 28 days after lesioning. No immunosuppression was used. Acellular collagen rolls served as controls. At 14, 28 and 90 days and 4 and 6 months after grafting, animals were analysed histologically with silver and Toluidine Blue stains and EM. The grafts often filled the lesion and the host borders they apposed exhibited only limited astrogliosis. By 14 days, bundles of unmyelinated and occasional thinly myelinated axons populated the periphery of Schwann cell implants. By 28 days and thereafter, numerous unmyelinated and myelinated axons were present in most grafts. Silver staining revealed sprouted axons at the implant border at 28 days and long bundles of axons within the implant at 90 days. Photographs of entire 1 micron plastic cross-sections of nine grafted areas were assembled into montages to count the number of myelinated axons at the graft midpoint; the number of myelinated axons ranged from 517-3214. Electron microscopy of implants showed typical Schwann cell ensheathment and myelination, increased myelin thickness by 90 days, and a preponderance of unmyelinated over myelinated axons. Random EM sampling of five Schwann cell grafts showed that the ratio of unmyelinated to myelinated axons was highest (20:1) at 28 days. These ratios implied that axons numbered in the thousands at the graft midpoint. Dissociated Schwann cells without matrix promoted axonal ingrowth and longitudinal orientation as effectively as did elongated Schwann cells accompanied by matrix. There was a suggestion that axonal ingrowth was at least as successful, if not more so, when the delay between lesioning and grafting was 28 rather than 5 days. Acellular collagen grafts did not contain axons at 28 days, the only interval assessed. In sum, grafts of Schwann cells in a rolled collagen layer filled the lesion and were well tolerated by the host. The Schwann cells stimulated rapid and abundant growth of axons into grafts and they ensheathed and myelinated these axons in the normal manner.

Anti-beta 1 integrin antibody inhibits Schwann cell myelination.

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypassed by adding a purified basal lamina component, laminin, to SC/neuron co-cultures. We have examined the role of laminin receptors, namely, the beta 1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the beta 1 subunit in association with the alpha 1 or alpha 6 subunit. In co-cultures of myelinating SCs, alpha 1 beta 1 is no longer present, alpha 6 beta 1 is still present but at reduced levels, and alpha 6 beta 4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that beta 1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of beta 1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-beta 1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an alpha 1 beta 1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the beta 1 subfamily of integrins other than alpha 1 beta 1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell.

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism.

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.