The asialoglycoprotein receptor (AsGR) is responsible for the selective endocytosis of galactose/N-acetyl galactosamine-terminating glycoproteins by hepatocytes. Expression of AsGR is characteristic of fully differentiated hepatocytes and is decreased or abolished in numerous examples of altered physiology and pathology of the liver. In contrast, increase in AsGR expression has been demonstrated only during biotin development and pregnancy. While the entire amino acid sequence of AsGR has been elucidated, the structure of the biologically active molecule differs due to post-translational modifications. In addition to previously demonstrated glycosylation and phosphorylation, recent data suggests that the receptor is fatty acid acylated. The long-term objectives of our studies are to understand the underlying biochemical mechanisms which regulate hepatocellular differentiation in response to changes in liver cell physiology.
The specific aims of this project are designed to define the kinetics of biotin induction of AsGR synthesis by a combination of metabolic labelling and quantitative immunoprecipitation. A proposed role for cyclic nucleotides as intermediates in this regulator's pathway will be studied using nucleotide analogs and the quantitation of their intracellular levels in response to biotin. Other plasma membrane proteins which are regulated by this mechanism will be identified using selective cell-surface labelling and 2D electrophoresis. The molecular level of protein synthesis responsive to biotin induction will be established by quantitation of AsGR mRNA by cDNA hybridization and the extent of its translation using polysome analysis. The proposed studies of AsGR induction and repression which occurs during murine pregnancy will compare the receptors biosynthetic rates to its mRNA concentrations to define the molecular level at which AsGR expression is being regulated. The new finding that AsGR is fatty acylated will be extended by determining the nature of the fatty acids, the type of acyl bonds and the peptide acyl site. Both metabolic and selective chemical labelling will be used in conjugation with HPLC analysis, selective hydrolysis and peptide sequencing. A potential role for acylation in the cellular compartmentalization of AsGR will be tested.
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