Project Details

Description

Human interleukin-3 (IL-3) is a pluripotent colony stimulating factor (CSF)
which has a crucial role in the development of early hematopoietic
progenitor cells and a role in some forms of acute lymphocytic leukemia.
In order to understand the mechanism of action of IL-3 in normal human
hematopoiesis as well as in leukemogenesis it is important to obtain a full
biochemical and molecular analysis of the IL-3 receptor and its associated
transducing mechanism in normal hematopoietic cells. This application
proposes to obtain such a characterization including the cloning of a
cDNA(s) for this receptor in a normal human hematopoietic cell. Although
the receptor appears in low abundance on human monocytes, the recent
availability of expression cloning techniques has made it possible to clone
such low abundance proteins. A cDNA library has been constructed from
human monocytes in a high efficiency animal cell expression vector (pcDNA1)
and pools of these clones will be transfected into COS-1 cells. The
transfected cells will be screened for [125I] IL-3 binding. After
identifying the cDNA clone coding for the IL-3 receptor the DNA sequence of
the clone will be determined and compared to sequences of known genes to
determine homologies. The affinity constant of the receptor transfected
into COS cells will be compared to that of the natural receptor. The
application also proposes to use polymerase chain reaction cDNA
amplification as a supplement to expression cloning. If the expression
cloning is successful this technique can also be used to identify unique
unidentified but related family members to the IL-3 receptor in human
monocytes. Amino acid sequence for the IL-3 receptor and the GM-CSF
receptor beta subunit deduced from the cDNA sequence, will be used to
synthesize peptides for production of monospecific polyclonal antibodies in
rabbits. The ability of these antibodies to precipitate the natural and
recombinant proteins will be assessed using [35S] methonine labelled cells.
When we have an antibody that is successful in precipitating the [35S]
methonine labelled IL-3 receptor we will also attempt to precipitate the
receptor bound to IL-3. This experiment should reveal whether or not the
binding of IL-3 to its receptor induces the association of any accessory
proteins with the IL-3 receptor. Finally, based on preliminary data
indicating a role for protein kinase C in IL-3 signal transduction, the
application proposes to examine the relationship of protein kinase C to IL-
3 mediated signal transduction. Examination of protein phosphorylation and
phospholipid hydrolysis in response to the stimulation of human monocytes
by IL-3 will be conducted. Taken together these experiments should
dramatically increase our understanding of the mechanism of action of human
IL-3, and should suggest further experiments to identify molecular
mechanisms fundamental to the regulation of normal human hematopoiesis and
leukemogenesis.Description
StatusFinished
Effective start/end date2/1/929/14/01

Funding

  • National Institutes of Health
  • National Institutes of Health: $21,395.00
  • National Institutes of Health
  • National Institutes of Health: $199,017.00

ASJC

  • Medicine(all)

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