Wheadon, Helen; (1997) Expression and function of the granulocyte-macrophage colony-stimulating factor receptor durning myeloid differentiation. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The receptor for the human Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF R) is composed of two subunits, an unique a chain which binds GM-CSF with low affinity and a [beta] chain, common to the interleukin-3 (IL-3) and IL-5 receptors, which does not bind GM-CSF on its own, but when associated with the [alpha] chain confers higher affinity binding. Scatchard analysis of primary human haemopoietic cells suggests that there are low, intermediate and high affinity classes of the GM-CSF R. To investigate the molecular basis of this, transfected NIH3T3 cells constitutively expressing the human GM-CSF R [beta] chain and inducibly expressing the human GM-CSF R [alpha] chain were generated. When cells were partially induced to express the a chain, the [alpha]:[beta] ratio was approximately 1:1 and Scatchard analysis revealed a single class of intermediate affinity receptors (Kd = 614 [plus-minus] 88 pM). In cells with fully induced a chain expression, the [alpha];[beta] ratio was approximately 3:1 and there was a switch to a dual high and low affinity receptor with Kd s of 67 [plus-minus] 32 pM and 1.7 [plus-minus] 0.56 nM respectively. This change was not associated with changes in [alpha]:[beta] stoichiometry as detected by cross-linking studies. Both the high and intermediate affinity receptors were able to activate the STAT 5 and the MAP kinase pathways, although there was a difference in the ligand dose response curves which was compatible with the different affinities. GM-CSF has different functional effects on immature and mature myeloid cells. In order to investigate these diverse effects and whether the early signalling events induced by GM-CSF are differentiation dependent, GM-CSF R expression and GM-CSF mediated activation of the JAK 2- STAT 5 and MAP kinase pathways were investigated using: HL60 cells induced to differentiate with dimethyl sulfoxide or retinoic acid, and purified CD34-t- cells induced to proliferate and differentiate with a combination of cytokines. GM-CSF stimulated MAP kinase activation in both the undifferentiated and differentiated HL60 cells. The maximum levels of activation occurred at 5 minutes and were broadly similar in both cell types. There was however a marked difference in the kinetics of activation, with the response being transient in the undifferentiated cells and disappearing by 15 minutes, whereas it persisted for at least 60 minutes in differentiated cells. GM-CSF mediated activation of STAT 5 was markedly upregulated (15-20 fold) by differentiation in HL60 cells but the kinetics of activation did not change. This was not due to a change in total cellular expression of STAT 5 but correlated with increased JAK 2 protein levels. Freshly purified CD34-I- cells expressed 36 ± 1 high affinity GM-CSF R per cell and the level of expression rose 5- 10 fold by day 8 in culture. The day 0 CD34+ cells were hyporesponsive to GM-CSF but by day 3, the cells were starting to proliferate and maximal JAK 2-STAT 5 and MAP kinase activation was induced. Further culture of the CD34-1- cells was however associated with prolongation of MAP kinase but not JAK 2-STAT 5 activation. These data indicate that differentiation modulates the activation of signalling molecules downstream from the GM-CSF R.

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