Ryan M. Drenan

FKBP12-Rapamycin-associated Protein or Mammalian Target of Rapamycin (FRAP/mTOR) Localization in the Endoplasmic Reticulum and the Golgi Apparatus

FKBP12-rapamycin-associated protein (FRAP) or mammalian target of rapamycin (mTOR) and its effector proteins form a critical signaling pathway that regulates eukaryotic cell growth and proliferation. Although the protein components in this pathway have begun to be identified, little is known about their subcellular localization or the physiological significance of their localization. By immunofluorescence, we find that both endogenous and recombinant FRAP/mTOR proteins show localization predominantly in the endoplasmic reticulum (ER) and the Golgi apparatus. Consistent with this finding, FRAP/mTOR is cofractionated with calnexin, an ER marker protein. Biochemical characterization suggests that FRAP/mTOR is a peripheral ER/Golgi protein with tight membrane association. Finally, we have identified domains of FRAP/mTOR which may mediate its association with the ER and the Golgi apparatus.

Palmitoylation regulates plasma membrane–nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family

The RGS7 (R7) family of RGS proteins bound to the divergent Gβ subunit Gβ5 is a crucial regulator of G protein–coupled receptor (GPCR) signaling in the visual and nervous systems. Here, we identify R7BP, a novel neuronally expressed protein that binds R7–Gβ5 complexes and shuttles them between the plasma membrane and nucleus. Regional expression of R7BP, Gβ5, and R7 isoforms in brain is highly coincident. R7BP is palmitoylated near its COOH terminus, which targets the protein to the plasma membrane. Depalmitoylation of R7BP translocates R7BP–R7–Gβ5 complexes from the plasma membrane to the nucleus. Compared with nonpalmitoylated R7BP, palmitoylated R7BP greatly augments the ability of RGS7 to attenuate GPCR-mediated G protein–regulated inward rectifying potassium channel activation. Thus, by controlling plasma membrane nuclear–shuttling of R7BP–R7–Gβ5 complexes, reversible palmitoylation of R7BP provides a novel mechanism that regulates GPCR signaling and potentially transduces signals directly from the plasma membrane to the nucleus.

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3–Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site‐specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Ü and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin‐mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Cholinergic Modulation of Locomotion and Striatal Dopamine Release is Mediated by α6α4* Nicotinic Acetylcholine Receptors

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express α6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive α6L9′S* receptors. α6L9′S mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by α6α4* pentamers, as α6L9′S mice lacking α4 subunits displayed essentially normal behavior. In α6L9′S mice, receptor numbers are normal, but loss of α4 subunits leads to fewer and less sensitive α6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires α4 subunits, implicating α6α4β2* nAChRs in α6L9′S mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by α6L9′S nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in α6L9′S, but not WT or α4KO/α6L9′S, mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from α6L9′S presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate α6α4β2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing α4, α6, β2, and β3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing α6 and β3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged β3 subunits were fully functional compared with receptors with untagged β3 subunits. We find that β3- and α6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent α4 and β2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of β3 and α6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single β3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for α4- and α6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of α6- and β3-containing receptors using TIRF.

The FKBP12‐rapamycin‐associated protein (FRAP) is a CLIP‐170 kinase

CLIP‐170/Restin belongs to a family of conserved microtubule (MT)‐associated proteins, which are important for MT organization and functions. CLIP‐170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP‐170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12‐rapamycin‐associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP‐170. CLIP‐170 is phosphorylated in vivo at multiple sites, including rapamycin‐sensitive and ‐insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin‐sensitive sites. In addition, rapamycin inhibited the ability of CLIP‐170 to bind to MTs. Our observations suggest that multiple CLIP‐170 kinases are involved in positive and negative control of CLIP‐170, and FRAP is a CLIP‐170 kinase positively regulating the MT‐binding behavior of CLIP‐170.

Structural differences determine the relative selectivity of nicotinic compounds for native α4β2^*-, α6β2^*-, α3β4^*- and α7-nicotine acetylcholine receptors

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR.

R7BP Augments the Function of RGS7·Gβ5 Complexes by a Plasma Membrane-targeting Mechanism

The RGS7 (R7) family of G protein regulators, Gβ5, and R7BP form heterotrimeric complexes that potently regulate the kinetics of G protein-coupled receptor signaling. Reversible palmitoylation of R7BP regulates plasma membrane/nuclear shuttling of R7·Gβ5·R7BP heterotrimers. Here we have investigated mechanisms whereby R7BP controls the function of the R7 family. We show that unpalmitoylated R7BP undergoes nuclear/cytoplasmic shuttling and that a C-terminal polybasic motif proximal to the palmitoylation acceptor sites of R7BP mediates nuclear localization, palmitoylation, and plasma membrane targeting. These results suggest a novel mechanism whereby palmitoyltransferases and nuclear import receptors both utilize the C-terminal domain of R7BP to determine the trafficking fate of R7·Gβ5·R7BP heterotrimers. Analogous mechanisms may regulate other signaling proteins whose distribution between the plasma membrane and nucleus is controlled by palmitoylation. Lastly, we show that cytoplasmic RGS7·Gβ5·R7BP heterotrimers and RGS7·Gβ5 heterodimers are equivalently inefficient regulators of G protein-coupled receptor signaling relative to plasma membrane-bound heterotrimers bearing palmitoylated R7BP. Therefore, R7BP augments the function of the complex by a palmitoylation-regulated plasma membrane-targeting mechanism.

Differential Expression and Function of Nicotinic Acetylcholine Receptors in Subdivisions of Medial Habenula

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotines aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and β4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, β2, β3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 μm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit.

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.