The G protein coupled 5-HTiA receptor has been implicated in regulating blood pressure and has been shown to improve hemodynamics during hypovolemic shock. It regulates many intracellular signaling cascades by binding to its endogenous ligand, serotonin (5-hydroxytryptamine, 5-HT). Despite the clinically relevant and diverse effects of the receptor, and the complexity of it signaling cascades, there is little mechanistic information available about key structural aspects associated with its activation and dynamic regulation. In that regard, previous studies have reported that the 5-HT1A receptor has calmodulin (CaM) binding domains in the third intracellular loop (i3L) of the receptor (2). Indeed, many GPCRs possess predicted potential CaM-binding domains. Unfortunately, little is known about the roles that direct binding of CaM may have on receptor function. Therefore, it is important to determine whether these CaM binding domains regulate signaling pathways through the 5-HT1A receptor. In addition, it is important to explore whether CaM binding to the 5-HTiA receptor plays a role in the relationship between structure and function of the 5-HT1A receptor, which may further our understanding of how this receptor is dynamically regulated.
Two aims will address those gaps in the knowledge base.
Specific Aim 1 : Determine the effects of CaM binding to the 5-HTiA receptor, on the generation and termination of 5-HT1A receptor signals. We will construct and characterize a CaM-binding deficient 5-HT1A receptor by making point mutations in the CaM binding region of the receptor. (1) We will characterize the G protein binding and activation stimulated by this receptor and compare with effects of the wild-type receptor. This will be accomplished by co-immunoprecipitation of the receptor and G protein a-subunits, by measuring GTPase activity, and GTPyS binding. (2) We will study the effects of the mutant receptor on the inhibition of adenylyl cyclase, phosphorylation of ERK, and activation of NHE-1. (3) We will determine whether CaM facilitates formation of a signaling complex between the receptor and key signaling enzymes. (4) We will study the role of CaM binding to the receptor in desensitization of the receptor.
Specific Aim 2. Determine the role of electrostatic interactions in CaM-mediated 5-HTiA receptor function and signaling.
This aim will begin a foray into structural analysis of the 5-HT1A receptor with reference to the role of CaM in maintaining receptor conformation. We will determine whether CaM-binding to the receptor alters receptor interactions with negatively charged phospholipids in the plasma membrane. We will use lipid vesicle and spectroscopic methods to compare the interactions of wild-type and mutated CaM-binding domains of the 5-HT1A with phospholipids in the plasma membrane. These studies will lay the foundation for future NMR studies and the generation of structural models to account for the effects of CaM on receptor activation and conformation.
|Coaxum, Sonya D; Blanton, Mary G; Joyner, Alisha et al. (2014) Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger. Am J Physiol Cell Physiol 307:C554-60|