Joav Prives - US grants

The goal of this project is to identify regulatory mechanisms controlling the expression of muscle surface proteins that mediate transmembrane signalling. Our studies will focus on the nicotinic acetylcholine receptor (AChR), a well characterized transmembrane oligomeric glycoprotein that functions as a ligand gated ion channel. During muscle differentiation and innervation, AChR undergoes marked changes in biogenesis and cell surface distribution. Using cultured embryonic muscle cells, we will study the regulation of AChR biogenesis and topogenesis, with emphasis on the contribution of cellular protein phosphorylation mechanisms. The studies to be performed are aimed at defining the significance of AChR phosphorylation to the regulation of AChR subunit assembly, as well as to the cell surface distribution and attachment to the cytoskeleton of oligomeric AChR. The assembly of AChR subunits will be measured by metabolic labeling and immunoprecipitation techniques, and AChR phosphorylation in situ will be assayed by the incorporation of (32P)i into immunoprecipitable AChR peptides, using pharmacological agents that alter cellular phosphorylation activities. The cell surface distribution of AChR will be monitored by fluorescence microscopy of cells labeled with a fluorescent ligand of AChR. Microinjection methods, and muscle cells transformed with Rous sarcoma viral mutants will be utilized to investigate the contribution of protein phosphorylation to AChR cell surface distribution. The attachment of AChR with cytoskeletal structures will be measured by mild detergent fractionation of muscle monolayers. It is anticipated that these studies will identify the regulatory significance of phosphorylation in AChR expression, and elucidate the role of post-translational modifications in the regulation of surface properties of specialized membrane ionic channel proteins.

IBN-9723145 Joav Prives PI SUMMARY These studies are designed to analyze the means by which muscle cells regulate the intracellular assembly and delivery to the cell surface of a neurotransmitter receptor, the nicotinic acetylcholine receptor (AChR). These receptors are crucial components for transmission of impulses across the synapses between motor neurons and muscle cells. Located on the subsynaptic surface membrane of muscle cells, AChRs function as acetylcholine-gated ion conducting membrane channels at the neuromuscular junction. Like genetically related receptors, each AChR is a complex comprised of five separate membrane spanning protein subunits. The molecular mechanisms by which AChR is assembled from its subunits is still undefined, as are the pathways that target AChR to restricted cell surface domains at postsynaptic regions of the muscle membrane. Elucidation of these mechanisms will lead to a more fundamental understanding of the regulation of signaling across synapses. The intracellular mechanisms that direct the folding of newly made subunits and their subsequent assembly into pentameric AChRs will be studied in cultured muscle cells that are genetically programmed for efficient expression of these receptors on the cell surface. An alternative experimental system - nonmuscle cells that have been transfected with the AChR subunits - will be utilized for the dissection of key regulatory aspects of the assembly process. Planned experiments are based on recent findings that AChR subunits are folded and assembled in an intracellular compartment termed the endoplasmic reticulum (ER) with the participation of calnexin, an ER resident molecular chaperone that interacts with newly synthesized polypeptide chains to prevent their aggregation and misfolding, and possibly to mediate oligomerization. The proposed studies are aimed at identifying the structural features that determine calnexin binding to AChR subunits and the m olecular mechanisms by which calnexin facilitates AChR assembly. The concentration of AChR into high density patches in the muscle membrane region directly adjacent to nerve endings is crucial for efficient neuromuscular transmission of impulses. Like the intracellular assembly of AChR, the formation and maintenance of high density surface AChR clusters under nerve endings requires highly regulated interactions between AChR and other proteins. The regulation of the surface distribution of AChR will be studied in muscle cell cultures, using an experimental approach that combines biochemical analysis of protein - protein interactions and high resolution immunofluorescence microscopy. These studies will focus on defining those signaling pathways that direct the redistribution of AChR into high density patches in response to neuronal cues. By focusing on the mechanisms that control AChR assembly and surface topography, the proposed studies should provide important insights into the regulatory pathways that modulate the functional state of synapses. Moreover, because several of the fundamental characteristics elucidated in the case of AChR have proven applicable to other members of the ligand-gated ion channel family, AChR studies will be valuable toward understanding the molecular basis of communication between excitable cells across synapses.