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Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.

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The development of erythroid progenitor cells is triggered via the expression of the erythropoietin receptor (EPOR) and its activation by erythropoietin. The function of the resulting receptor complex depends critically on the presence of activated JAK2, and the complex contains a large number of signaling molecules recruited to eight phosphorylated tyrosine residues. Studies using mutant receptor forms have demonstrated that truncated receptors lacking all tyrosines are able to support red blood cell development with low efficiency, whereas add-back mutants containing either Tyr 343 or Tyr 479 reconstitute EPOR signaling and erythropoiesis in vivo. To study the contribution of tyrosines to receptor function, we analyzed the activation of essential signaling pathways and early gene induction promoted by different receptor structural variants using human epidermal growth factor receptor/murine EPOR hybrids. In our experiments, receptors lacking all tyrosine residues or the JAK2-binding site did not induce mitogenic and anti-apoptotic signaling, whereas add-back mutant receptors containing single tyrosine residues (Try 343 and Tyr 479 ) supported the activation of these functions efficiently. Profiling of early gene expression using cDNA array hybridization revealed that (i) the high redundancy in the activation of signaling pathways is continued at the level of transcription; (ii) the expression of many genes targeted by the wild-type receptor is not supported by add-back mutants; and (iii) a small set of genes are exclusively induced by add-back receptors. We report the identification of several early genes that have not been implicated in the EPOR-dependent response so far. Erythropoietin (EPO),2 the prime growth factor regulating the erythroid lineage, induces the proliferation of immature red blood cells and their differentiation into mature erythrocytes (1). The corresponding EPO receptor (EPOR), a member of the type 1 cytokine receptor family, is expressed on the surface of erythroid burst-and colony-forming units (2). In contrast to other receptor types, these receptors lack any intrinsic activity and are activated by the induction of a special conformation of pre-existing homodimers after ligand binding (3). The recruitment and activation of the cytosolic tyrosine kinase JAK2 have been shown to be the first and crucial steps in the formation of a multiprotein receptor complex that is responsible for the initiation of diverse signaling pathways (4). Activated JAK2 mediates the phosphorylation of tyrosine residues within the intracellular receptor domain, providing docking sites for Src homology 2 (SH2) domain-encoding effector molecules, including the JAK2 substrate STAT5, a transcription factor that is rapidly translocated to the nucleus (5). Among additional proteins that have been shown to be physically associated with the EPOR following ligand binding are phospholipase C␥1 (6) and phosphatidylinositol 3-kinase (7). Furthermore, negative regulators are also engaged and terminate receptor-dependent signa...

Currently, primary osteoporosis is the most frequent metabolic disease in women after menopause [1]. The resulting loss of bone mass is accompanied by an increased risk of skeletal fragility. One reason for the development of osteoporosis might be an impaired function of mature osteoblasts. To evaluate the involvement of specific growth factors in bone remodeling, cell cultures of osteoblastic cells derived from nonosteoporotic and osteoporotic postmenopausal women were established. The influences of TGF beta-1 and IGF-I on proliferation and mRNA expression of TGF beta-1 were investigated by [3H]-thymidine incorporation and competitive RT-PCR. We found IGF-I to have no significant effect on proliferation in cells of osteoporotic and nonosteoporotic patients. In contrast, differences were found in TGF beta-1 mRNA expression after application of IGF-I. Application of TGF beta-1 enhanced its own mRNA expression in both groups in a similar manner. Whereas the proliferation of cells of nonosteoporotic patients was inhibited by (10(-10) M) TGF beta-1, this treatment led to an increased proliferation of cells of osteoporotic patients.