Receptor-mediated Intracellular Transport Research Articles

Skeletal muscle expression of clathrin and mannose 6‐phosphate receptor in experimental chloroquine‐induced myopathy

Previous studies suggest that the muscle fiber lysosome system plays a central role in the increased formation of autophagosomes and autolysosomes that occurs in the context of chloroquine-induced myopathy. The goal of this study was to characterize the contribution of receptor-mediated intracellular transport, particularly the endosomal pathway, to the abnormal accumulation of vacuoles in experimental chloroquine myopathy. Expression of the mannose 6-phosphate receptor (M6PR) and clathrin were analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Accumulation of vacuoles was observed only in chloroquine-treated denervated muscles. Further, clathrin immunostaining and M6PR messenger ribonucleic acid (mRNA) were significantly increased in denervated soleus muscle from saline- and chloroquine-treated rats compared to contralateral, innervated muscles. However, there was no difference in clathrin levels when comparing saline- and chloroquine-treated denervated muscles. These data suggest that chloroquine activates the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus (an endosomal pathway) as well as autophagosome formation (an autophagic process) in skeletal muscles. Vacuoles may subsequently accumulate secondary to abnormal formation or turnover of autolysosomes at or after fusion of autophagosomes with early endosomes.

Expression of lysosome-related proteins and genes in the skeletal muscles of inclusion body myositis.

Despite the unknown etiology and pathogenesis of sporadic inclusion body myositis (s-IBM), investigators have speculated that the lysosome system in muscle fiber plays a central role in rimmed vacuole formation, a hallmark of s-IBM. We explored the role of receptor-mediated intracellular transport and autophagy in the lysosomal system in the abnormal accumulation of rimmed vacuoles in s-IBM. Expressions of mannose 6-phosphate receptor (M6PR), clathrin and hApg5 and hApg12 were analyzed in muscle biopsy specimens from patients with s-IBM, amyotrophic lateral sclerosis (ALS) or peripheral neuropathy and in normal human muscle specimens by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Most muscle fibers in control specimens showed little or no immunoreactivity for clathrin and M6PR, which are involved in the receptor-mediated intracellular transport. Abnormal increases in both proteins were observed mainly in the sarcoplasm of atrophic fibers in all diseased specimens. In s-IBM muscles in particular, clathrin and M6PR were often observed inside rimmed vacuoles and in the sarcoplasm of vacuolated or non-vacuolated fibers. mRNA levels of hApg5 and hApg12, which are involved in autophagic vacuole formation, as well as of M6PR and clathrin were significantly increased in s-IBM muscles in comparison to levels in normal and ALS/peripheral neuropathy muscles. Our results suggest that the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus and autophagic vacuole formation (i.e., autophagy) in the lysosome system are activated in s-IBM muscles. Remarkable accumulation of rimmed vacuoles is thought to occur because of abnormal lysosome function, especially the formation or turnover of autolysosomes after the fusion of autophagic vacuoles with the early endosomes or because of the increase in the rate of muscle fiber breakdown.

Effect of chloroquine-induced myopathy on rat soleus muscle sarcoplasm and expression of clathrin.

Clathrin-coated vesicles are involved in receptor-mediated intracellular transport pathways related to lysosomal proteolysis. Clathrin levels were significantly elevated in denervated soleus muscles from chloroquine- and saline-treated rats as compared with their contralateral, innervated muscles. No difference was found in the clathrin levels of the denervated muscles in both groups. The accumulation of autophagic vacuoles was marked only in chloroquine-treated muscles after denervation. These findings suggest that chloroquine does not inhibit intracellular trafficking of clathrin-coated vesicles during the overdevelopment of autophagic vacuoles.

Clathrin: its role in receptor-mediated vesicular transport and specialized functions in neurons.

Clathrin constitutes the coat of vesicles involved in three receptor-mediated intracellular transport pathways; the export of aggregated material from the trans-Golgi network for regulated secretion, the transfer of lysosomal hydrolases from the trans-Golgi network to lysosomes and receptor-mediated endocytosis at the plasma membrane. The clathrin subunits and the other major coat constituents, the adaptor polypeptides, interact in specific ways to build the characteristic polygonal clathrin lattice and to attach the coat to integral membrane receptors. Both clathrin coat assembly and disassembly on the cytoplasmic side of the membrane are multistep processes that are regulated by the coat constituents themselves and by cytosolic proteins and factors. Neurons represent a cell type with distinct morphology and special demands on exocytic and endocytic pathways that requires neuron-specific constituents and modifications of clathrin-coated vesicles.

Binding receptors for alpha-L-fucosidase in human B-lymphoid cell lines.

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of alpha-L- fucosidase in lymphoid cells. The binding of alpha-L-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding alpha-L-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.

Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen.

The third external domain of major histocompatibility class I antigens has a highly conserved disulfide bridge between cysteine-203 and cysteine-259. To elucidate the functional significance of this disulfide bridge, we have produced a mutant H-2Ld gene by site-directed mutagenesis in which the codon for cysteine-203 is changed to a codon for serine, which is unable to form a disulfide bridge. The mutant H-2Ld gene was introduced into mouse L cells and its expression has been studied. No measurable expression of the H-2Ld antigen was detected on the cell surface of the transformants by antibody-binding assays. However, a large quantity of the mutant H-2Ld antigen was found in the cytoplasm of the transformants as observed by immunoprecipitation of metabolically labeled lysate and by immunocytochemistry of membrane-permeabilized cells, using an antibody specific for the first external domain of the H-2Ld antigen. The mutant antigen was glycosylated and associated, at least in part, with beta 2-microglobulin. Subcellular fractionation experiments indicated that the transport of the antigen was blocked between the endoplasmic reticulum and the plasma membrane. It is concluded that structural integrity of the third external domain is a prerequisite for intracellular transport of class I antigens. On the basis of these findings we suggest that the domain structure containing the disulfide bridge serves as a signal structure necessary for receptor-mediated intracellular transport and that this requirement is the evolutionary basis for high conservation of similar structures present throughout the immunoglobulin supergene family.

Receptor-mediated transport of IgG across the intestinal epithelium of the neonatal rat.

The absorptive epithelium of the neonatal rat is developmentally specialized to transfer maternal immunoglobulin G (IgG) intact to the circulation while other milk protein are digested. The epithelial cells of the duodenum and proximal jejunum which are responsible for IgG transfer represent a particular striking experimental model for study of receptor-mediated intracellular transport. Receptors located on the luminal plasma membrane selectively bind the Fc region of IgG. The IgG enters the cell by constitutive endocytosis within coated vesicles and is then released at the basolateral plasma membrane. Morphological evidence supports a model in which IgG crosses the cell as a ligand-receptor complex that dissociates only on exposure to a pH 7.4 environment found at the basolateral cell surface. Although uptake of IgG at the luminal plasma membrane is highly selective, small but significant amounts of other proteins enter the cell apparently non-selectively. Nevertheless, these latter proteins are not transferred across the cell. Double-tracer experiments indicate that IgG and these other proteins enter the cell simultaneously within the same endocytic vesicles, but that non-membrane-bound proteins are removed from the IgG transport pathway by an as yet poorly defined mechanism and sequestered within small apical vacuoles and lysosomes.