Role of Homer proteins in mast cells.

Mast and basophile cells are important players in the response to allergic reactions. This response is triggered by a sustained elevation in cytosolic Ca2+ levels, mediated by the CRAC1 current and its key player, the STIM1-Orai1 complex, which activates degranulation and hence allergic response. The Homer protein family are scaffold proteins that modulate store-operated Ca2+ signalling while the role of this family in mast cells is not known. In this project I aim to elucidate the function of Homer isoforms in store-operated calcium entry, CRAC currents and secretion of mast cells. In my preliminary experiments, I detected that the long isoforms of Homer1, b/c, are endogenously expressed in RBL-2H3 mast cells and associate with STIM1 in a Ca2+-dependent manner. Molecular biology will be used to design different mutants of Homer1 and STIM1 to characterize their binding domain/s with a combination of biochemical as well as confocal FRET microscopy methods. Previously, I have demonstrated that impairment in the association between STIM1-Homer1 isoforms diminishes the formation of the STIM1-Orai1 complex, thus modifying SOCE in human platelets. By using siRNA specific against each Homer1 isoform, I will determine which isoform/s is involved in the CRAC complex formation of mast cells. My preliminary results further demonstrated that interference in Homer1 proteins directly n/re-activation profile of CRAC currents in RBL-2H3 mast cells. Therefore I will use fura-2 Ca2+ imaging and the patch-clamp technique together with confocal FRET microscopy to reveal the choreography of Homer proteins in the STIM1-Orai1 complex in the endogenous as well as over-expressed CRAC currents in RBL and HEK-293 cells respectively. Finally, I will characterize the role of Homer1 proteins in CRAC-induced granule release in mast cells with siRNA interference as well as Homer mutants. In summary these investigation will reveal insight into the role of Homer proteins in STIM1-Orai1 mediated Ca2+ entry, CRAC currents and secretion in allergic response in mast cells.

Several diseases commonly name as first world diseases, as diabetes mellitus or atherosclerosis, are leading cause of death in western industrialized countries. Furthermore, new studies and researches are demonstrating daily that several cancers types and, even the aging diseases, like Alzheimer or Parkinson, present some common characteristics. In most of these diseases the calcium dynamics are altered. Thus, the studies focused on the mechanisms that tune the calcium homeostasis of cells key to the understanding of such diseases. Within this project, granted by the Austrian Science Fund (FWF), we focused on various aspects of Ca2+ release activated Ca2+ (CRAC) channel regulation. Human cells receive and generate a variety of chemical and molecular signals: to proliferate, release substances or migrate into a tissue. One of the main signals is calcium. In many cells, this calcium influx is mediated via the CRAC channel complex composed of two key proteins, STIM1 and Orai, the communication of which and its modulation by additional proteins and lipids have been examined in my research project.