The objective of this proposal is to determine the biochemical basis for high and low affrnity interleukin-2 (IL-2) receptor expression on human lymphocytes. The high affinity receptor binds IL-2 with a Kd of 3-10 pM, is internalized in response to ligand, and appears to mediate the proliferative effects of IL-2. The low affinity receptor, whose function has yet to be elucidated, binds IL-2 with a Kd of lnM, and comprises 90% of the receptors present on activated T cells and T cell lines. The IL-2 receptor, which migrates on SDS-PAGE as a broad band of Mr 50-55,000, has been purified, cloned, and expressed. The protein backbone of 33,000 daltons undergoes complex post-translational modification, including addition of N-linked and O-linked sugars, sialic acid, and phosphate. The intracytoplasmic domain of the receptor contains only 13 amino acids, insufficient to provide a mechanism for ligand-induced signal transduction, such as a protein kinase activity so common to other growth factor receptors. Moreover, the IL-2 receptor gene produces 12 distinct mRNA transcripts, which differ in the use of 2 promoters, 3 polyadenylation sites, and a spliceable element. The functional correlates of these gene products have remained elusive. Despite extensive knowledge about this receptor, we do not understand the structural basis, regulation, or function of these two species of receptors. Preliminary investigation suggests a number of potential mechanisms to explain differences in affinity, and provides insight into the mechanism of signal transduction. In the first part of this study, we will evaluate culture conditions which influence the expression of high and low affinity receptors, and generate cell populations which preferentially express one or the other. We will then subject these populations to rigorous biochemical examination, testing the hypotheses that differences in receptor affinity are due to a) the presence of a second IL-2 binding protein, b) an auxillary protein, which forms a receptor complex conferring high affinity, c) post-translational modification by differential glycosylation, or protein phosphorylation, or d) quanine nucleotide binding proteins. In the third part of this study, we will employ these cells to test the hypothesis that different mRNAs correlate with high or low affinity receptors. The investigation of the structural basis of high and low affinity receptor expression is likely to yield new knowledge about how IL-2 and its receptor may regulate immune responsiveness.
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