Project Summary/Abstract: Receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and cell adhesion molecules (CAMs) are three major families of cell surface proteins in all eukaryotic cells, and together represent the primary first responders for cells to respond an extracellular stimulus and initiates a variety of signaling pathways to subsequently regulate cell proliferation and differentiation, promote cell survival, and modulate cellular metabolism and cell-to-cell communication. Mutations affecting these signaling pathways result in many human syndromes and diseases, such as various types of neurodegenerative disorders and cancer. The clinical importance of these signaling proteins has motivated the development of targeted therapies designed to block the activation of the membrane receptors and the downstream signal transduction. Increasing evidence has suggested that there is significant signaling crosstalk between these three membrane protein families at the plasma membrane level and these proteins can form highly organized membrane micro- or nano-clusters with unique biochemical and biophysical properties, dictating the signaling outcome. However, the molecular mechanisms by which how such crosstalk and compartmentalization of membrane-associated signaling proteins are initiated to modulate the sensitivity and specificity of the downstream signaling remain largely elusive. Our recent discovery of a newly identified actin-spectrin-based membrane-associated periodic skeleton (MPS) structure being a signaling platform for RTK transactivation by GPCRs and CAMs in neurons provides molecular insights into how the cooperative action among these cell surface proteins can be coordinated to give rise to the downstream signaling. The objective of this proposal is to combine super-resolution imaging, cell and molecular biology tools, and mass spectrometry analyses, to investigate the detailed molecular mechanisms responsible for the MPS-mediated cell signaling, by identifying the key molecular interactions responsible for the MPS-dependent initiation of the signaling protein clustering (i.e., complex formation) and examining the roles of liquid-liquid phase separation, receptor endocytosis, and contact sites between the plasma membrane and intracellular membrane-bound intracellular organelles in the MPS-mediated signaling. Our proposed research will not only broaden our fundamental understanding of cell signal transduction controlled by the membrane skeleton and the phase separation behaviors of signaling proteins, but also help suggest new drug targets for human diseases including neurodegenerative diseases and cancer.