The main purpose of these studies will be to analyze in detail the regions of the growth hormone receptor (GHR) and the JAK2 kinase that are activated in response to growth hormone binding, to identify signaling molecules that bind to these sequences, and to attempt to determine which signalling pathways are activated by these signalling molecules. The grant is divided into five specific aims. 1. Mass spectrometroscopic analyses of the proteolytic cleavage products of the GH receptor will be used in combination with phosphopeptide mapping and mutagenesis of the GHR to determine which of the tyrosines in GHR are phosphorylated. 2. The principal investigator will utilize the same methodology as in specific aim 1 to gain insight into which tyrosines within JAK2 are phosphorylated in response to GH. 3. The investigator will examine which tyrosines and/or regions of GHR in JAK2 are required for the binding of specific signalling molecules and the activation of discrete signalling pathways. The initial emphasis will be given to the signalling molecules STAT, SHC, IRS-1 and IRS-2. 4. The investigator will determine whether there are any cellular actions of GH that are independent of JAK2. 5. Experiments using available antibodies as well as the yeast 2 hybrid system will attempt to identify previously undiscovered signalling molecules that interact with GHR or JAK2. An alternative approach will be to screen a lambda expression library with phosphorylated peptides derived from GHR or JAK2 to identify such proteins.
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