John Raymond Hepler - US grants

DESCRIPTION (Adapted from Investigator's Abstract): Many hormones and neurotransmitters rely on the Gq class of heterotrimeric G proteins (Gqalpha, G11alpha, G14alpha, and G16alpha) and the inositol lipid signaling pathway to exert their actions at target tissues. RGS proteins (Regulators of G protein Signaling) are a newly discovered family (17 isoforms) of proteins that completely block G protein functions and serve a central role in regulating hormone receptor and G protein signaling. Very little is known about the broad cellular actions of RGS proteins and how their functions are regulated. The PI's recent studies provide the first evidence that one RGS protein found in brain, RGS4, directly inhibits the functions of Gqalpha and abolishes receptor and G protein directed inositol lipid signaling in cell membranes. Preliminary studies also reveal that RGS4, an intrinsically hydrophilic protein, is localized at the plasma membrane in mammalian cells. These observations suggest mechanistic questions regarding RGS4 interactions with the Gqalpha signaling pathway. The PI has generated a broad spectrum of experimental tools for studying RGS proteins and G proteins that will allow him to define cellular and biochemical mechanisms underlying RGS4/Gqalpha interactions. By combining biochemical, cellular, and molecular approaches, the major goals of these studies will be to: 1. Determine whether RGS4 can regulate the functions of G11alpha, G14alpha and G16alpha as well as Gqalpha. 2. Define the molecular basis for RGS4 membrane association and identify underlying biochemical factors and their roles in RGS4 function and inositol lipid signaling. 3. Determine whether activation of Gqalpha-linked signaling pathways regulates the cellular level of endogenous RGS4 and other RGS proteins to inhibit Gqalpha signaling. These experiments will define molecular mechanisms that dictate RGS and G protein interactions, and offer new insight into regulation of receptor signaling. RGS proteins are newly appreciated and important regulators of G protein signaling, and their actions may underlie poorly understood human disease states and/or contribute to the onset of tolerance to clinical agents that stimulate G protein signaling pathways. Information gained from these studies will help us better understand RGS proteins as potential therapeutic targets.

Many hormones and neurotransmitters rely on the Gq class of heterotrimeric G proteins (Gq/11alpha, G14alpha, and G15/16alpha) to exert their actions at target issues. Gqalpha family members activate PLCbeta and inositol lipid signaling, and established models suggest that the cellular actions of these Galpha are identical and result from activation of Ca PKC pathways. However compelling evidence now indicated that Gq/11alpha, G14alpha, and G15/16alpha differ markedly in their overall cellular responses, their interactions with certain protein binding partners, and their cellular and biochemical properties. Contrary to established models, my working hypothesis is that Gq/11alpha, G14alpha and G15/16alpha regulate multiple overlapping and distinct signaling cascades, and are regulated differently by host cells to elicit a distinct profile of cellular responses. Consistent with this idea, Gqalph family members are expressed in different cells, and Gqalpha interacts with multiple signaling proteins besides PLCbeta, though the relative contribution of each binding partner to Gqalpha signaling is unknown. Furthermore, Gq/11alpha, G14alpha and G15/16alpha share only 57 percent overall amino acid identity, and just 12 percent identity within their first 35 residues (N- terminus) which contains fatty acids that are essential for regulating Galpha signaling capacity. Although the acylation state of G14alpha and G15/16alpha is unknown, sequence alignments predict that Gqalpha family members are modified differently. Using modem molecular, cellular, and biochemical approaches study G protein functions, the specific aims will be to: 1. Define biochemical modifications present on Gq/11alpha, G14alpha, and G15/16alpha important for regulating signaling functions. 2. Define factors that regulate Gq/11alpha, G14alpha and G15/16alpha signaling capacity including target subcelluar localization and interactions with protein binding partners. 3. Determine the diversity of cell signaling responses elicited by Gqalpha, G14alpha and G15/16alpha in selected cells, and the relative contribution of Galpha binding partners to those responses. Heterotrimeric G proteins serve essential roles in cell physiology by inking cell surface receptors to intracellular responses. G protein dysfunction is the direct cause of a growing list of human diseases, and the majority of currently available drugs exert their actions on G protein signaling pathways. These experiments will determine functional correlates for the biochemical differences observed among the Gqalpha family of G proteins, and offer new insights into the diversity and regulation of signaling responses elicited by these important G proteins. Information gained from these studies will help us to better understand G proteins as therapeutic targets.

DESCRIPTION (provided by applicant): Neurotransmitters and hormones rely upon G proteins to exert their effects on target cells. In contrast, growth factor tyrosine kinase receptors recruit a cascade of linked signaling proteins including ras-like GTPases to regulate cell growth and differentiation. Recent compelling evidence indicates that additional novel signaling proteins, including certain regulators of G protein signaling (RGS proteins) integrate G protein and ras superfamily-directed pathways at many levels. RGS proteins are highly diverse, multifunctional proteins that bind directly to G proteins to regulate their functions. RGS14 contains domains (RGS, GoLoco and RID domains) that we and others have shown to interact with active Galpha-i /o-GTP (RGS domain), inactive Galpha-i/o-GDP (GoLoco domain) and with the ras-like GTPases rapl and rap2 (RID domain). Activation of rap1/2 pathways in host cells stimulates MAPK/Erk kinase signaling and neuronal differentiation. Roles for RGS14 as a dual regulator of Gai and Gao signaling are not known. Furthermore, how RGS14, Galpha-i/o and rap1/2 interact to regulate one anothers functions, and what cellular mechanisms are involved in regulating these interactions and linked cellular responses is not known. Our preliminary studies indicate that RGS14 is phosphorylated by kinases involved in G protein and rap signaling. Our hypothesis is that RGS14 is an integrator of G protein (Galpha-i/o) and rap-directed signaling pathways, and that stimulus-initiated phosphorylation regulates RGS 14 interactions with Galpha and rap, its subcellular localization, and its signaling functions. Using modern molecular, cellular and biochemical approaches, the specific aims will be to: 1. Determine roles for RGS 14 as a bifunctional regulator of receptor and Galpha-i and Galpha-o signaling in membranes. 2. Define cellular and biochemical mechanisms that regulate RGS 14 membrane recruitment and attachment, subcellular localization, and association with mChoR, Galpha-i/o, and rap1/2 in host B35 cells. 3. Define cellular mechanisms for regulating RGS14 phosphorylation in cells, and roles for phosphorylation on RGS14 interactions with mChoR/Gi/o and rap1/2 in vitro, and linked signaling pathways in B35 cells. 4. Determine the effects of overexpressing RGS14 or eliminating native RGS14 in B35 cells on mChoR/Gi/o and rap1/2- directed signaling events and cellular responses. Impact: These studies will identify previously unrecognized cell signaling roles for RGS proteins, and provide important information about novel mechanisms for cross-talk between G protein signaling pathways and rap/MAPkinase signaling pathways. Findings from these studies will clarify our understanding of the complexity of G protein/RGS protein signaling, and identify potential new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): Neurotransmitters and hormones rely upon G proteins to exert their effects on target cells. In contrast, growth factor tyrosine kinase receptors recruit a cascade of linked signaling proteins including ras-like GTPases to regulate cell growth and differentiation. Recent compelling evidence indicates that additional novel signaling proteins, including certain regulators of G protein signaling (RGS proteins) integrate G protein and ras superfamily-directed pathways at many levels. RGS proteins are highly diverse, multifunctional proteins that bind directly to G proteins to regulate their functions. RGS14 contains domains (RGS, GoLoco and RID domains) that we and others have shown to interact with active Galpha-i /o-GTP (RGS domain), inactive Galpha-i/o-GDP (GoLoco domain) and with the ras-like GTPases rapl and rap2 (RID domain). Activation of rap1/2 pathways in host cells stimulates MAPK/Erk kinase signaling and neuronal differentiation. Roles for RGS14 as a dual regulator of Gai and Gao signaling are not known. Furthermore, how RGS14, Galpha-i/o and rap1/2 interact to regulate one anothers functions, and what cellular mechanisms are involved in regulating these interactions and linked cellular responses is not known. Our preliminary studies indicate that RGS14 is phosphorylated by kinases involved in G protein and rap signaling. Our hypothesis is that RGS14 is an integrator of G protein (Galpha-i/o) and rap-directed signaling pathways, and that stimulus-initiated phosphorylation regulates RGS 14 interactions with Galpha and rap, its subcellular localization, and its signaling functions. Using modern molecular, cellular and biochemical approaches, the specific aims will be to: 1. Determine roles for RGS 14 as a bifunctional regulator of receptor and Galpha-i and Galpha-o signaling in membranes. 2. Define cellular and biochemical mechanisms that regulate RGS 14 membrane recruitment and attachment, subcellular localization, and association with mChoR, Galpha-i/o, and rap1/2 in host B35 cells. 3. Define cellular mechanisms for regulating RGS14 phosphorylation in cells, and roles for phosphorylation on RGS14 interactions with mChoR/Gi/o and rap1/2 in vitro, and linked signaling pathways in B35 cells. 4. Determine the effects of overexpressing RGS14 or eliminating native RGS14 in B35 cells on mChoR/Gi/o and rap1/2- directed signaling events and cellular responses. Impact: These studies will identify previously unrecognized cell signaling roles for RGS proteins, and provide important information about novel mechanisms for cross-talk between G protein signaling pathways and rap/MAPkinase signaling pathways. Findings from these studies will clarify our understanding of the complexity of G protein/RGS protein signaling, and identify potential new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): Recent studies with gene knock-out mice indicate a prominent role for the functionally linked signaling proteins RGS2, Gq-alpha (Gqa), alpha-1A-adrenergic (a1A-AR) and m1 muscarininc cholinergic receptors (M1 AChR) in regulation of vascular hypertension, cardiac hypertrophy, sympathetic control of cardiac function, and vascular tone. However, molecular models for how these proteins interact are not well understood. RGS proteins bind directly to activated Ga subunits to modulate and integrate their functions. Very little is known about how RGS selectivity for target Ga is determined in cells. Recent studies suggest that RGS and GPCR are functionally linked in cells but direct interactions have not been shown. Based on these observations, we tested whether RGS proteins and GPCR interact directly. We found that RGS2 (but not RGS1 or RGS16) binds directly and selectively to the third intracellular loop (i3) of the Gq/11-linked M1AChR and a1A-AR, but not i3 of a1B-AR or a1D-AR or Gi/o-linked M2- or M4AChR. Our studies show that RGS2, M1-13, and Gqa form a stable heterotrimer complex, and that the N-terminus of RGS2 is responsible for RGS binding to the i3 of both receptors. My working hypothesis is that RGS proteins form stable, functional complexes with preferred GPCR to selectively modulate the signaling functions of those receptors and linked G proteins. Using molecular, cellular and biochemical approaches, the Specific Aims will be to: Aim 1: Identify amino acids on the M1AChR, a1A-AR and RGS2 responsible for direct binding. Aim 2: Determine roles for RGS2 on receptor/ligand binding and functional Gq/11 a coupling. Aim 3: Determine the effects of RGS2 on GRK2 binding/phosphorylation and arrestin binding to receptors on their desensitization, internalization, and intracellular trafficking. Aim 4: Determine the effects of suppressing native RGS2 mRNA/protein on native M1AChR and a1A-AR signaling functions in cells/VSM tissues that natively expresses a1A-AR or M1AChR and RGS2. These studies will define novel cellular mechanisms for regulating neurotransmitter and hormone signaling, and identify potential new molecular targets for therapeutic intervention in cardiovascular diseases.

DESCRIPTION (provided by applicant): We recently discovered that RGS14 is a novel mediator of hippocampal-based learning/memory. We find that RGS14 is expressed predominantly in brain, specifically within dendrites/neurites of hippocampal neurons important for learning/memory (i.e. pyramidal cells of the CA1/CA2 region). Our novel mice lacking the RGS14 gene/protein (RGS14-KO) exhibit a marked enhancement of spatial learning/memory and novel-object recognition and of long-term potentiation (LTP) of postsynaptic neurotransmission in CA1 neurons. These findings strongly suggest that RGS14 regulates signaling events critically important for hippocampal-based learning/memory. However, the molecular and cellular actions of RGS14 in hippocampal neurons remain poorly defined. We find that RGS14 binds Ric8A, an unconventional guanine- nucleotide exchange factor activator for G1i, and also to inactive Gi11/3-GDP, active H-Ras and Rap2 and their effectors the Raf kinases. In cells, RGS14 co-localizes with its binding partners at the plasma membrane to inhibit stimulated Ras/Raf/Erk signaling. When complexed with its partners in cells, RGS14 is phosphorylated at unknown site(s) with unknown functional consequences. Of note, RGS14 partners H-Ras, Rap2, Gi and linked Erk pathways regulate synaptic plasticity associated with learning/memory in hippocampal neurons including neurite outgrowth, dendritic remodeling, and trafficking of glutamate receptors. My working hypothesis is that RGS14 is a tightly regulated scaffolding protein that integrates unconventional Gi and H-Ras/Raf/MAPkinase signaling events important for synaptic plasticity involved with learning, memory and cognition in the hippocampus. Our Specific Aims will be to: Aim 1. Determine the protein/protein interactions and molecular mechanisms by which RGS14 integrates unconventional Ric8A/Gi1 and H-Ras/Rap2/Raf kinase signaling. Aim 2: Determine sites on RGS14 that are phosphorylated when complexed with Gi11/3, H- Ras or Rap2 in cells, the involved kinases, and how phosphorylation impacts RGS14 signaling. Aim 3: Determine roles for RGS14 in regulating molecular and physical markers of H- Ras/Raf/MAPkinase-directed synaptic plasticity in hippocampal neurons. Aim 4: Determine roles for RGS14 in regulating postsynaptic neurotransmission and linked behaviors that result from H-Ras/Raf/Erk-directed synaptic plasticity in the hippocampus Rap/MAPkinase signaling events and the morphology of primary hippocampal neurons. Impact: These studies will provide key insight about RGS14 as a novel regulator and integrator of neurotransmitter signaling events that modulate neuronal plasticity, learning and memory. PUBLIC HEALTH RELEVANCE: These studies will define novel molecular changes and underlying mechanism that occur in brain cells during normal physiological processes like learning and memory, and pathological processes such as Alzheimer's disease, other neurodegenerative diseases and epilepsy/seizures.

DESCRIPTION (provided by applicant): We recently discovered that RGS14 is a novel suppressor of both synaptic plasticity/LTP in CA2 hippocampal neurons and hippocampal-based learning and memory. These studies are the first to implicate this gene/protein (RGS14) and this hippocampal region (CA2) in synaptic plasticity relating to learning and memory. Very little is known about the enigmatic CA2 region of brain, the resident genes there, or the underlying molecular mechanism(s) by which RGS14 regulates LTP there. We have found that RGS14 is a multifunctional scaffolding protein that integrates G protein and H-Ras/Raf/ERK signaling to inhibit certain forms of growth factor-directed MAP kinase signaling. RGS14 contains specific domains that bind active Gi/o1-GTP (RGS domain), inactive Gi11/2-GDP (GPR/GL domain) and active H-Ras/Rap2 and Raf (tandem Ras binding domains, or RBD). Several other signaling proteins and pathways have been identified in CA2 neurons that modulate LTP there, and that also link to RGS14 binding partners (Gi/o-linked A1 adenosine receptors, EGF and FGF Tyr-kinase receptors linked to H-Ras/Raf/MAP kinase signaling). My working hypothesis is that RGS14 integrates key signaling pathways that are critical for regulating synaptic plasticity in hippocampal CA2 neurons. The goal for these studies will be to develop experimental tools and to identify (for future study) molecular signaling pathways that RGS14 interfaces with in CA2 neurons to modulate synaptic plasticity. The Specific Aims will be to: Aim 1: Develop novel lentiviral delivery systems that will allow us to rescue lost RGS14 function (inhibition of LTP) in CA2 neurons, and to test if expression of RGS14 in CA1 neurons suppresses synaptic plasticity there. Aim 2: Develop novel lentiviruses encoding targeted loss-of-function mutations of RGS14 that will determine the role of the different RGS14 signaling partners/pathways in the regulation of CA2 synaptic plasticity/LTP. Aim 3: Determine how RGS14 interacts with known signaling mechanisms that regulate LTP in CA2 neurons. IMPACT: These studies will generate new experimental tools that will determine which of the identified signal functions of RGS14 are most important for its actions in CA2 neurons, (thereby helping to direct future studies), and establish what known pathways in CA2 neurons RGS14 interacts with to regulate synaptic plasticity important for key human cognitive functions. PUBLIC HEALTH RELEVANCE: These studies will define novel molecular mechanisms that underlie normal physiological processes such as learning and memory and social behaviors that are altered in human disease states such as schizophrenia or the autism and bipolar spectrum of disorders.

DESCRIPTION (provided by applicant): RGS14 is a newly appreciated effector protein that integrates G protein and H- Ras/Raf1/ERK signaling pathways. RGS14 is a brain protein that is highly enriched in and largely restricted in its expression pattern to dendrites and spines of neurons of hippocampal region CA2. We recently discovered that RGS14 is critically important as a natural suppressor of synaptic plasticity (LTP) in CA2 neurons. Furthermore, we show that ectopic expression of RGS14 in CA1 neurons where RGS14 is not expressed blocks LTP there, suggesting that RGS14 engages common cell signaling pathways critical for synaptic plasticity. Unlike the well-studied CA1 region, very little is known about CA2 neurons or RGS14 there. The CA2 is implicated in human neurological diseases including schizophrenia, the autism/bipolar spectrum of disorders, and epilepsy. Mice lacking RGS14 (RGS14-KO) exhibit a marked and unexpected enhancement in spatial learning and object recognition memory compared with wild type littermates, but show no differences in non- hippocampal-dependent behaviors. RGS14-KO mice also exhibit a surprisingly robust nascent LTP with enhanced neuronal excitability at glutamatergic synapses in CA2, with no impact on plasticity in adjacent CA1 neurons. Together, these findings highlight the importance of understanding the molecular mechanism(s) whereby RGS14 regulates neuronal/synaptic plasticity. Within CA2/CA1 neurons, LTP expression and its suppression is due to both Ca++-dependent (CaM, CaMKII) and Ca++-independent (ERK, cAMP/PKA) mechanisms. RGS14 binds inactive G?i1/3-GDP and active H-Ras-GTP to form a heterotrimeric signaling complex that integrates G protein and MAPK signaling pathways. RGS14 also binds calmodulin (CaM) in a Ca++-dependent manner. These findings suggest RGS14 is well positioned to regulate plasticity in host neurons. Consistent with this idea, the nascent LTP in CA2 neurons following loss of RGS14 is dependent on MEK/ERK signaling. Based on this, my working hypothesis is that the G?i-GDP:RGS14:H-Ras-GTP signaling complex integrates G protein, MAPK and Ca++/CaM signaling pathways to serve as a natural suppressor of synaptic plasticity in host neurons. However, the molecular/structural basis for how RGS14 binds G proteins, H-Ras and CaM to operate as a signaling switch/integrator is unknown. Furthermore, the dynamic subcellular localization and regulation of native RGS14 in its natural host CA2 neurons, and the signaling pathways that the G?i1:RGS14: H-Ras signaling complex engages to regulate synaptic plasticity in host CA2/CA1 is entirely unknown. The Specific Aims are: AIM 1. Determine the structural basis and interdomain dynamics for how RGS14, G?i, H-Ras and CaM interact to form a functional signaling complex. AIM 2: Determine the cellular properties of native RGS14 and how RGS14 engages the H-Ras/ERK signaling pathway to regulate synaptic plastic in natural host CA2 neurons. AIM 3: Determine the signaling pathways used by the G?i:RGS14: H- Ras complex in CA2 or CA1 hippocampal neurons to regulate LTP in hippocampal slice preparations.

? DESCRIPTION (provided by applicant): Recent high profile reports have identified the enigmatic hippocampal area CA2 as being at the center of an entirely new hippocampal circuit that is essential for social learning linked to autism and schizophrenia. Unlike the well-understood trisynaptic (dentate gyrus (DG)-CA3-CA1) hippocampal circuit, very little is known about area CA2. Previously, this brain region had been indirectly implicated in the autism/bipolar spectrum of disorders and schizophrenia. However, these new reports demonstrating CA2's essential role in social learning provide a compelling novel link between this brain region with autism/bipolar disorders and schizophrenia. Therefore, understanding signaling proteins and pathways in CA2 neurons offers an exciting new opportunity for the development of novel therapeutic tools to treat these devastating and vexing diseases. We have shown that the brain protein RGS14 is expressed in pyramidal neurons of hippocampal region CA2 in adult mouse and humans. RGS14 is an unusual scaffold/effector protein that integrates G protein, MAPkinase, and Ca++/CaM signaling pathways essential for synaptic plasticity. We recently discovered that RGS14 is critically important as a natural suppressor of synaptic plasticity in CA2 neurons but not CA1. Mice lacking RGS14 (RGS14-KO) exhibit a marked and unexpected enhancement of long-term potentiation (LTP) in CA2 neurons and in the acquisition of hippocampal-based spatial learning and memory, with no differences in other behaviors. These findings highlight the importance of understanding the molecular mechanism(s) whereby RGS14 regulates synaptic plasticity in its natural host CA2 hippocampal neurons. To date, very little is known about signaling proteins/pathways in CA2 neurons. Furthermore, our understanding of RGS14 signaling functions is based largely on binding interactions of recombinant RGS14 and partners in non-native expression systems. Based on these findings, our working hypothesis is that RGS14 is a central nexus that regulates signaling pathways essential for synaptic function and plasticity. Yet nothing is known about natural binding partners of native RGS14, and the signaling pathways it engages within its natural host CA2 neurons. Consistent with this idea, our preliminary studies show that native RGS14 exists as a high molecular weight protein complex and binds unidentified partners from brain. The goal of these studies is to define novel signaling pathways in RGS14-expressing CA2 hippocampal neurons for future study, and the specific signaling pathways that native RGS14 engages to regulate synaptic plasticity. AIM 1: Determine the cell transcriptome and proteome of RGS14-expressing CA2 hippocampal neurons and CA2-specific signaling pathways using bioinfomatics analysis. AIM 2: Identify natural binding partners of native RGS14 from brain (wild type vs RGS14-KO). AIM 3: Determine roles for newly identified CA2-specific receptor pathways in regulating LTP in CA2 neurons of hippocampal slice preparations from wild type and RGS14-KO mice.

Recent reports identified hippocampal area CA2 as the center of a new hippocampal synaptic circuit essential for social learning, linked to autism and schizophrenia. Unlike the classic dentate gyrus-CA3-CA1 hippocampal circuit, little is known about area CA2. New reports of CA2's role in social learning provide a compelling link between this brain region and these psychiatric diseases. Therefore, understanding signaling proteins and mechanisms in hippocampal neurons is key to understanding their roles in human neuro-pathophysiology. We reported that RGS14 is highly enriched in CA2 pyramidal neurons of adult mouse, monkey and humans. We reported that RGS14 is a scaffolding protein that integrates Gi, ERK, and Ca++ signaling pathways essential for synaptic plasticity. Moreover, we discovered that RGS14 is critically important as a natural suppressor of synaptic plasticity in CA2 neurons but not CA1. RGS14-KO mice exhibit a marked and unexpected reinstatement of long-term potentiation (LTP) in CA2 neurons paired with an enhancement of hippocampal- based learning and memory, with no differences in other behaviors. RGS14 also blocks LTP in CA1 neurons when introduced, indicating it engages signaling pathways common to both CA2 and CA1 neurons. These findings highlight the importance of understanding cellular mechanism(s) whereby RGS14 regulates synaptic plasticity in hippocampal neurons. While most of what we know about RGS14 relates to regulation of postsynaptic signaling (Gi, ERK and Ca++) at dendritic spines, we also found that RGS14 localizes to various subcellular compartments including the nucleus. Within monkey brain, we show a subpopulation of native RGS14 localizes to the nuclei of neurons. Consistent with this, RGS14 contains both a nuclear localization sequence (NLS) and export sequence (NES) that enables it to shuttle in-out of the nucleus. Remarkably, we identified rare human variants of RGS14 (L504Q, R507Q) that alter the NES site resulting in RGS14 accumulation in the nucleus. This observation, along with the fact that RGS14 is a natural nucleo-cytoplasmic shuttling protein, beg the question: what is RGS14 doing in the nuclei of neurons? Our studies here show that native RGS14 localizes to the nuclei of host cells in close proximity to open DNA and active RNA Polymerase II. Based on this, we hypothesize that RGS14 serves a key role in the nuclei of hippocampal neurons, possibly as a synapse- to-nucleus co-regulator of transcribed genes important for synaptic plasticity. Therefore, the goals of these exploratory studies are to: AIM 1: Determine the effects of RGS14 and nuclear localizing human variants of RGS14 on synaptic plasticity in hippocampal neurons. AIM 2: Identify RGS14 binding partners and their signaling roles in the nuclei of hippocampal neurons. AIM 3: Determine the effects of RGS14 and nuclear localizing RGS14 variants on gene transcription in hippocampal neurons. !

SUMMARY: RGS14 is a multifunctional signaling protein that integrates G protein, MAPkinase, and calcium/CaM signaling pathways. RGS14 is found in brain where it is highly enriched in dendrites and spines of pyramidal neurons in hippocampal region CA2. We discovered that RGS14 is critically important as a natural suppressor of synaptic plasticity (long-term potentiation, LTP) in CA2 neurons. Our studies show that ectopic delivery of RGS14 to CA1 neurons where RGS14 is not expressed blocks LTP there, suggesting that RGS14 engages common signaling pathways that are critical for synaptic plasticity in both populations of neurons. Unlike CA1 neurons, little is known about CA2 neurons where RGS14 is expressed. This enigmatic brain region has been implicated in social behavior and human neuro-psychological diseases including schizophrenia, the autism/bipolar disorders, and epilepsy. Remarkably, we have found that mice lacking RGS14 (RGS14-KO) exhibit an unexpected enhancement of spatial learning and object recognition memory compared with wild type littermates, with no differences in non-hippocampal-dependent behaviors. Furthermore, RGS14-KO mice expressed a surprisingly robust nascent LTP with enhanced neuronal excitability at glutamatergic synapses in CA2, with no impact on plasticity in adjacent CA1 neurons. Together, these findings highlight the importance of understanding the molecular mechanism(s) whereby RGS14 regulates LTP and synaptic plasticity within CA2 hippocampal neurons. LTP and associated spine plasticity depends on a rise in postsynaptic calcium due to glutamate activation of NMDA/GluN channels and the voltage-gated calcium channel Cav1.2, which result in activation of CaM and CaMKII signaling pathways. These pathways, in turn, increase actomyosin-driven trafficking and insertion of AMPA/GluA receptor vesicles at the synapse that result in increased spine size (i.e. structural plasticity). Of note, we find that RGS14 suppresses the activity-induced rise in spine calcium, inhibits Cav1.2, binds Ca++/CaM, and is phosphorylated by CaMKII. Furthermore, we find that RGS14 suppresses spine structural plasticity associated with LTP, and exists in brain as part of a high-molecular weight complex enriched with spine myosins (MyoV, MyoVI, MyoII) and actin binding proteins. Based on these observations, our working hypothesis is that RGS14 suppresses spine calcium by inhibiting Cav1.2 channels, and blocks LTP by engaging the actomyosin system (in a regulated way) to limit surface AMPA receptors. We further propose that these actions of RGS14 are regulated by its binding partners CaM, CaMKII, H-Ras/Rap2-GTP and Gai1. To test these ideas, we propose the following experiments. AIM 1. Determine how Ca++/CaM binding and CaMKII phosphorylation modulate established RGS14 functions. AIM 2: Determine how RGS14 regulates Cav1.2 and suppresses postsynaptic calcium signaling in hippocampal neurons. AIM 3: Determine how RGS14 impacts AMPA receptor recycling and engages the actomyosin system to suppress spine plasticity in hippocampal neurons.

PROJECT SUMMARY: PTH and FGF23 initiate signaling pathways in renal proximal tubule cells that converge on the [NPT2A:NHERF1] complex to inhibit phosphate uptake. Mice lacking the PDZ protein NHERF1 and humans harboring NHERF1 mutations are hypophosphatemic. Important gaps exist in understanding the elements involved in these actions due to unidentified factors. Numerous GWAS studies of patients with chronic kidney disease implicate RGS14, which harbors a PDZ-recognition motif. We propose that RGS14 is a novel regulator of hormone-sensitive phosphate transport, and provide preliminary data to support this idea. RGS14 is scaffold that integrates G protein, MAPK, and Ca++/CaM signaling pathways. Its actions are best understood in rodent brain, where RGS14 suppresses synaptic plasticity and hippocampal-based learning. Nothing is known about RGS14 effects on hormone action or in kidney. Our preliminary findings show that RGS14 suppresses both PTH- and FGF23-regulated phosphate transport. RGS14 persistently attenuates PTH-regulated phosphate transport in primary human proximal tubule cells. RGS14 knock-down by siRNA reverses this action to reveal full PTH activity. RGS14 add-back to cells lacking the protein blocks PTH actions. Human RGS14 contains a C- terminal PDZ ligand (-DSAL) that directly binds NHERF1. Mutations in the PDZ-ligand disrupt RGS14 binding to NHERF1 and interfere with RGS14 regulation of hormone actions. These findings identify RGS14 as an entirely new element regulating hormone action on a vital homeostatic activity, and raise the hypothesis that RGS14 is a novel regulator of hormone-sensitive [NPT2A:NHERF1]-mediated phosphate transport in renal proximal tubule cells. Three Aims will test this premise. AIM 1 will define how RGS14 regulates assembly, disassembly of [NPT2A:NHERF1], its internalization and hormone-sensitive phosphate uptake. AIM 2 will define the locus of RGS14 effects on PTH and FGF23 signaling and the impact of hormone-directed NHERF1 phosphorylation on RGS14 regulation of the [NPT2A:NHERF1] complex. AIM 3: will define how PTH- and FGF23-directed signaling affect RGS14 interactions with NHERF1 and the [NHERF1:NPT2A] complex. These studies will identify novel roles and molecular mechanisms by which RGS14 regulates NPT2A function and hormone-sensitive phosphate transport in kidney as related to kidney disease.