REGULATION OF THE INSULIN RECEPTOR KINASE

Project: Research project

Project Details

Description

The overall objectives of this research are focused at understanding the
molecular mechanisms by which extracellular insulin binding to the Alpha
subunit of the insulin receptor results in the transmembrane activation of
the intracellular Beta subunit-protein kinase-domain. Initially we plan to
use the purified human placental insulin receptor to examine the role of
subunit-subunit interaction in the insulin-dependent activation of the
insulin receptor Beta subunit autophosphorylation and exogenous substrate
protein kinase activities. This will be accomplished by the careful
analyses of the functional properties, both insulin binding and protein
kinase activities, of an isolated AlphaBeta heterodimeric insulin receptor
complex from the purified Alpha2Beta2 heterotetrameric complex. These
AlphaBeta heterodimeric complexes will then be used to determine the
functional symmetry of the Alpha2Beta2 heterotetrameric complex in terms of
the spatial relationship between the insulin binding site(s) and the Beta
subunit autophosphorylation as well as ATP binding sites. These studies
will then be extended to determine if one intracellular Beta subunit can
function as a competitive inhibitor for the exogeneous protein kinase
activity of the other Beta subunit in an analogous fashion to the
regulatory subunit of the cAMP-dependent protein kinase.

Recent preliminary results from our laboratory have suggested that ATP may
be an allosteric regulator of the insulin receptor protein kinase
activity. This will be evaluated by examining the kinetic properties of
ATP and various ATP analogous (i.e.: AMPPNP) on the insulin receptor
protein kinase activity as well as in direct substrate and nuleotide
binding experiments. Similar studies will then be performed on the
isolated AlphaBeta heterodimeric complexes which presumably should contain
only one insulin and one ATP binding site. We also plan to directly
determine which reduced cysteine residues are critical for the insulin
receptor protein kinase activity and which may be responsible for the
activation of the insulin receptor protein kinase by the previously
predicted insulin induced intramolecular disulfide-sulfhydryl
rearrangement. These studies will employ (3H)NEM labeling followed by
proteolytic peptide mapping and microsequencing. Further, these studies
will be extended to determine which of the possible disulfide bonds are
responsible for the covalent association between the Alpha and Beta
subunits as well as between the AlphaBeta heterodimeric complexes that are
required for the formation of the mature Alpha2Beta2 complex.
StatusFinished
Effective start/end date1/1/901/1/90

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases

ASJC

  • Molecular Biology

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