EXCEED THE SPACE PROVIDED. Allergens stimulate release of mast cell inflammatory mediators by crosslinking IgE bound to the high affinity IgE receptor, Fc_RI. By immunogold electron microscopy of native membrane sheets, we have recently shown that multiple, distinct microdomains mediate signaling and receptor internalization in mast cells. Receptors cluster together with Lyn in resting cells, are phosphorylated by Lyn very rapidly after stimulation in the signal initiation step, and then segregate from Lyn as receptors accumulate in osmiophilic regions of membrane. For signal propagation, these dark patches of membrane also accumulate Syk, PLC?2, p85, Gab2 and Grb2, identifying the osmiophilic patch as a primary site of Fc_RI signaling. Recruitment of AP-2, Eps15 and clathrin to receptors in osmiophilic patches, and the budding of clathrin-coated vesicles there, also define it as the primary site of Fc_RI internalization. Secondary signaling domains include p85 and PLC_,I and may be organized around the palmitoylated transmembrane adaptor protein, LAT. We hypothesize that these distinct microdomains perform separate tasks in mast cell signaling and receptor trafficking. To do so, they require unique lipid and protein compositions. We further propose that, after the initial segregation of receptors from Lyn, receptors go through a series of interaction and segregation steps with signaling and internalization machinery. The 'segregation model' predicts that these successive association/dissociation steps are ordered in part by features of the osmiophilic patch. To test these hypotheses, we propose to use a combination of electron microscopy, recombinant protein expression, biochemical assays and mass spectrometry to map the distribution of proteins and lipids in primary and secondary domains. We will test roles for acylation or prenylation of key signaling proteins in their targeting to either primary or secondary domains. We will also define the requirement for ubiquitin l/gases and multiubiquitination in receptor endocytosis. This work will define roles for microdomains in the processes of signal initiation, signal propagation and receptor internalization. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051575-03
Application #
6841949
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Minnicozzi, Michael
Project Start
2003-02-01
Project End
2008-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
3
Fiscal Year
2005
Total Cost
$282,099
Indirect Cost
Name
University of New Mexico
Department
Pathology
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
DeVore, M S; Stich, D G; Keller, A M et al. (2015) Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging. Rev Sci Instrum 86:126102
Mahajan, Avanika; Barua, Dipak; Cutler, Patrick et al. (2014) Optimal aggregation of Fc?RI with a structurally defined trivalent ligand overrides negative regulation driven by phosphatases. ACS Chem Biol 9:1508-19
Cleyrat, Cédric; Darehshouri, Anza; Anderson, Karen L et al. (2013) The architectural relationship of components controlling mast cell endocytosis. J Cell Sci 126:4913-25
Pullen, Nicholas A; Barnstein, Brian O; Falanga, Yves T et al. (2012) Novel mechanism for Fc{epsilon}RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production. J Biol Chem 287:2045-54
Espinoza, Flor A; Oliver, Janet M; Wilson, Bridget S et al. (2012) Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins. Bull Math Biol 74:190-211
Carroll-Portillo, Amanda; Surviladze, Zurab; Cambi, Alessandra et al. (2012) Mast cell synapses and exosomes: membrane contacts for information exchange. Front Immunol 3:46
Espinoza, Flor A; Wester, Michael J; Oliver, Janet M et al. (2012) Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor Fc?RI of mast cells. Bull Math Biol 74:1857-911
Wilson, Bridget S; Oliver, Janet M; Lidke, Diane S (2011) Spatio-temporal signaling in mast cells. Adv Exp Med Biol 716:91-106
Nowak-Lovato, K L; Wilson, Bridget S; Rector, Kirk D (2010) SERS nanosensors that report pH of endocytic compartments during Fc?RI transit. Anal Bioanal Chem 398:2019-29
Wells, Nathan P; Lessard, Guillaume A; Goodwin, Peter M et al. (2010) Time-resolved three-dimensional molecular tracking in live cells. Nano Lett 10:4732-7

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