MOLECULAR STRUCTURE OF ENDOSOMES IN DEVELOPING INTESTINE

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During development, the absorptive cells of the intestine go through a stage in which they are highly specialized for the endocytosis of macromolecules from the intestinal lumen. During this time (which, depending upon the species, may occur in utero or after birth) these cells contain an elaborate endosomal complex. This complex has been suggested to be important in the selective transepithelial transport of growth factors that are crucial for the normal development of the intestine and other organ systems. However, little is known about the cellular mechanisms that control the selective transepithelial transport of macromolecules while preventing pathogens from opportunistically infecting through the same route. To identify the mechanisms that underlie selective transepithelial transport, it is necessary to characterize the compartment involved in sorting of internalized macromolecules. The goals of the current proposal are to obtain the cDNA sequence for an integral membrane glycoprotein (entubin), which is found in the endosomes of the developing intestine of several species, including human, but is absent from the mature intestine or any other cell type. A rat neonatal intestine cDNA library will be screened by hybridization using oligonucleotide probes generated after obtaining internal amino acid sequence from affinity purified entubin. In the rat, entubin is present in two electrophoretically distinguishable forms, indicating either alternative splicing of mRNAs, multiple genes, or differential glycosylation. Therefore, this cDNA will be used to characterize the number and size of transcriptional units encoding entubin and to determine the number of genes encoding entubin. In addition, the cDNA will be used to screen other epithelial cell and non- epithelial cell types for entubin-related proteins. If entubin related proteins are detected in other cell types, it is possible that entubin (or entubin-like proteins) are required for the normal function of endosomes. In addition, the carbohydrate composition of this glycoprotein will be determined by specific glycosidase digestion of affinity purified entubin and together with the deduced amino acid sequence used to predict the topology of the protein in the membrane. This characterization of entubin is a first step in understanding the molecular structure of endosomal membranes. Also, although the function of entubin is unknown, it provides a unique and valuable marker of the endosomal compartment. By transfecting cell lines with the cDNA for entubin it will be possible to carry out detailed morphological and biochemical studies of the targeting of endosomal proteins and to address the questions of biogenesis of endosomal compartments, endosomal dynamics, and recycling of endosomal components. In the long term, these studies will provide insight into the mechanism of sorting and targeting of developmentally important ligands such as growth factors.