A G Protein Coupled Nicotinic Receptor: How the Alpha 7 Nicotinic Acetylcholine Receptor Regulates Hippocampal Axon Growth via an Interaction with G Proteins

Historically neurotransmitter receptor classes have been divided into two categories: ionotropic and metabotropic. Ionotropic receptors are typically multi-subunit structures that when activated conduct ions through a central pore, leading directly to the depolarization/hyperpolarization of the plasma membrane, generation of action potentials, and release of neurotransmitters from axon terminals. Metabotropic receptors on the other hand are single protein, seven transmembrane domain structures (though multi-subunit complexes like the GABAB receptor exist), which upon binding of a ligand, activate specialized sets of second messenger proteins such as the heterotrimeric guanosine nucleotide binding proteins (G proteins). These proteins upon activation translocate to other parts of the cell where they regulate a host of downstream signaling molecules such as adenylyl cyclase and phospholipase C, gene signaling cascades such as the ERK/MAPK pathway, and cytoskeletal regulatory pathways underlying neurite growth and development. In recent years a number of studies have come out demonstrating that these two receptor classes may not be as functionally distinct as was formerly thought.;A number of ionotropic receptors have already been shown to interact with G proteins, contributing to their signaling milieu. Examples include: 1) the &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which has been shown to interact with Galphai subunits for the purpose of regulating silent synapses, 2) the glycine receptor, which has been shown to interact with betagamma G protein subunits, thereby regulating channel opening and kinetics, and 3) the nicotinic acetylcholine receptors (nAChRs) alpha4beta2 and alpha3beta4alpha5, which have been shown to interact with both the Galpha;o and betagamma subunits. The consequence of these interactions has great promise for understanding cellular phenomena that remain mysterious to this day and for piecing together the intracellular protein pathways that will aid in the development of newer and better drugs in the fight against brain disease.;nAChRs are a pentameric class of ionotropic receptors belonging to the Cys-loop receptor family and found widely expressed throughout the brain. 11 different nAChR subunits have been identified in the mammalian brain (alpha2-alpha11 and beta2-beta4). Each subunit is composed of 4 transmembrane domains with a long intracellular loop formed between the 3rd and 4th transmembrane domain. This domain is where the majority of protein interactions with the receptor is hypothesized to occur and is the most variable, least conserved part of each nAChR subunit, conferring special properties to each receptor. alpha7 is unique among the nAChRs in that it possesses 5 identical alpha subunits and can conduct calcium at levels roughly ten times that of the other common nAChRs found throughout the brain. alpha7 has also been implicated as a regulator of neural development. A number of studies have demonstrated that alpha7 expression is critical for establishing the inhibitory GABA signal necessary for proper circuit formation. It has also been shown that alpha7 expression is paramount for glutamatergic synaptogenesis within the hippocampus. Less studied however is the role of alpha7 in regulating axon growth, perhaps the key starting point for neural development.;In this dissertation I demonstrate that alpha7 interacts with G proteins to regulate calcium influx and the cytoskeletal machinery underlying growth. This was accomplished through a variety of biochemical, proteomic, and live cell imaging techniques. The following work reveals that alpha7, acting in concert with the G proteins Galphao and Galphaq, regulate axon growth via intracellular calcium release from internal stores. These studies demonstrate a first ever alpha7 mediated calcium signal within the growth cone, an interaction with G proteins, and a mechanism by which alpha7 can regulate the cytoskeletal machinery in the developing neurite. These studies aid in understanding the critical role of iontropic neurotransmitter receptor interactions with G proteins, and in particular how metabotropic functions of the alpha7 receptor are instrumental in proper brain development.;In a parallel line of work I demonstrate a similar mechanism underlying alpha4beta2 nAChR signaling within immune cells in vivo. In that study I show that alpha4beta2 receptors, expressed in CD4+ T-cells, can regulate cell division, interleukin-6 (IL-6) release, and a Th2 immune response via an interaction with a Galphai/o protein pathway. This research is important for two reasons: 1) it provides evidence for an immunological function of nAChRs, widely found expressed in immune cells, but to date have not been detected electrophyiologically, suggesting that nAChR signaling within immune cells likely operates through a second messenger pathway; 2) nAChR interactions with second messenger pathways enables for better understanding of the effects of nicotine on immunity, serving as both a platform for biomarker discovery in smokers and a possible drug target for a host of immune diseases.