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The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels. [ABSTRACT FROM AUTHOR]

Explores the mechanism that allows adrenergic agonists to evoke secretion in equine sweat glands. Generation of second-messenger signals; Modulation of membrane chloride permeability; Formulation and application of specialized preparations of cultured sweat gland epithelial cells; Function of beta-adrenoceptors.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role., (Copyright © 2017 the American Physiological Society.)

The dopamine D 3 receptor (D 3 R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D 3 R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D 3 R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D 3 R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D 3 R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D 3 R dimerization.

Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the alpha(1b)-adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a "dominant-negative" in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal N-glycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the alpha(1b)-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.

The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine alpha(1a)-adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by beta-arrestin-dependent endocytosis. Internalization of the ligand did not occur in beta-arrestin-deficient cells; was blocked or reversed by another alpha(1) ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human alpha(1a)-AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with beta-arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in beta-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are beta-arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that alpha(1a)-ARs recycle rapidly by an agonist-independent, constitutive, beta-arrestin-dependent process and that this can transport "alpha-blockers" into cells carrying these receptors.