Dissection of the cytoplasmic domains of cytokine receptors involved in STAT and Ras dependent proliferation

Abstract

Cytokine receptors have di erent signaling requirements which ultimately lead to various physiological responses.In an e ort to precisely characterize the molecular determinants involved in the proliferative response mediated by cytokines, we examine dose-dependent proliferation of the bc (GM-CSF, IL-3, IL-5) and homodimeric (G-CSF, TPO) cytokine receptors. Here we report that all cytokine receptors tested activate mostly STAT3 and STAT5. While STAT3 had a positive e ect on bc cytokine receptor dependent proliferation, STAT5 was strongly inhibitory. Similarly, G-CSF and TPO lead to activation of STAT3 and STAT5 but, unlike the bc cytokine receptors, both stimulated cellular growth. On the other hand, Ras activation was necessary for all receptor mediated proliferation with the exception of G-CSF R. Truncated mutants of the receptors intracellular domains were used to delineate the functional domains involved in JAK/ STAT and Ras activation linked to cellular growth. For instance, we revealed a critical role for the speci®c alpha subunit of the bc receptors in triggering receptor activation, STAT3 stimulation and proliferation, while Ras activation originates from the distal intracellular portion of the bc subunit. Finally, we showed that proximal STAT activation is the triggering event of G-CSF and TPO receptor function.

β-arrestin 2 modulates the activity of nuclear receptor RAR β2 through activation of ERK2 kinase

“…Cells were thus transfected with RAR b2 and b-arrestin 2, along with constructs encoding dominant negative forms of various signaling proteins. The dominant negative properties and selectivities of these mutants have been documented elsewhere (Burstein et al, 1998;Piu et al, 2002). While some mutants affected the baseline activity of the assay, they did not significantly influence the agonist-dependent activity of the RAR b2 in response to AM-580 ( Figure 2a, see no drug data).…”

“…To assess this hypothesis, the functional assay R-SATt (Receptor Selection and Amplification Technology) assay was used, that allows to monitor proliferation of gene targets in a ligand-dependent fashion. Such assays have been developed for a number of receptor families, ranging from GPCRs (Brauner-Osborne and Brann, 1996) to RTKs (Burstein et al, 1998;F.Piu unpublished) and cytokine receptors (Piu et al, 2002). NIH-3T3 fibroblasts were transiently transfected with the individual RAR and RXR receptor subtype, in the presence or absence of b-arrestins.…”

“…Similarly, the b-arrestin 2 mutant AAEA was created. Other constructs have been described (Piu et al, 2002).…”

“…R-SATt assays R-SATt (Receptor Selection and Amplification Technology) is a proprietary cell-based functional assay that allows one to monitor receptor-dependent proliferative responses and has been described elsewhere (Piu et al, 2002). The technology has been validated for a number of receptors ranging from GPCRs (Brauner-Osborne and Brann, 1996), RTKs (Burstein et al, 1998) and cytokine receptors (Piu et al, 2002).…”

“…The technology has been validated for a number of receptors ranging from GPCRs (Brauner-Osborne and Brann, 1996), RTKs (Burstein et al, 1998) and cytokine receptors (Piu et al, 2002). Its principle resides in the genetic selection and amplification of the nuclear receptors in a ligand-dependent manner.…”