Ret Receptor: Functional Consequences of Oncogenic Rearrangements.
Annual rept. 15 Sep 95-14 Sep 96,
CALIFORNIA UNIV SAN DIEGO LA JOLLA
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Retptc2 is a soluble, constitutively active oncogene isolated from human papillary thyroid carcinomas. Sequence analysis of retptc2 indicated that the gene resulted from a reciprocal rearrangement event between the cAMP-dependent protein kinase regulatory subunit Ialpha RIalpha and the entire tyrosine kinase domain of the Ret receptor. Using the crystal structure of the insulin receptor tyrosine kinase, we have obtained a working model of the Retptc2 kinase domain. This model was used in conjunction with a microinjection assay and a yeast 2-hybrid screen to 1 determine that the RIalpha dimerization domain is critical for eliciting the Retptc2 mitogenic response, and 2 to identify a number of tyrosine residues of Retptc2 which are both autophosphorylated and interact with downstream signaling proteins containing src-homology domains such as Grb10, PLCgamma, and a LIM domain containing protein, Enigma. Peptide substrates containing these putative phosphorylated tyrosine residues were synthesized and tested in an in vitro kinase assay using Retptc2 expressed in human kidney 293 cells. We have expressed retptc2 in a acterial expression system to study and characterize the physical properties of the purified protein. In addition we have made chimeras of the epidermal growth factor receptor and insulin receptor which mimic he retptc2 gene to test whether the dimerization domain of RIalpha can activate other receptor tyrosine kinases.
- Medicine and Medical Research