TRANSMEMBRANE ACTIVATION OF THE INSULIN RECEPTOR KINASE

Project: Research project

Project Details

Description

The overall objectives of this research proposal 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 alpha beta
heterodimeric insulin receptor complex from the purified alpha2
beta2 heterotetrameric complex. These alpha beta heterodimeric
complexes will then be used to determine the functional
symmetry of the alpha2 beta2 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 exogenous protein kinase activity of the other beta subunit
in an analogous fashion to the regulatory subunit of the cAMP-
dependent 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 alpha beta
heterodimeric complexes which presumably should contain only
one insulin an 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 alpha beta heterodimeric complexes that are
required for the formation of the mature alpha2 beta2 complex.
StatusFinished
Effective start/end date1/1/906/30/90

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases

ASJC

  • Molecular Biology

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