Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β₂ Adrenergic Receptor at the Single-Molecule Level

G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environmentlipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes

On Solving the Initial Problem of LR Arrays

Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome <i>bo</i>₃ from <i>Escherichia coli</i>, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome <i>bo</i>₃, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions

Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (<i>Thermomyces lanuginosus Lipase</i>) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme’s major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

<div><p>Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient <i>Drosophila</i> and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the <i>Drosophila</i> brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a <i>Drosophila</i> model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.</p></div

Rescue of the phenotype of PICK1-deficient mice by GH administration.

<p>(A) Lean body mass was evaluated by MRI scanning before GH administration and subsequently once every week both in PICK1-deficient mice (black squares) and in saline-treated WT littermates (white circles). Before treatment the difference between the two groups was highly significant (**<i>p</i> = 0.0027), after a week the difference decreased but was still significant (*<i>p</i> = 0.050), and after 2 and 3 wk no significant difference were observed. (B) IGF-1 mRNA level in the liver as determined by RT-PCR was significantly lower in PICK1-deficient mice compared to untreated mice (*<i>p</i> = 0.0045), however this difference was not observed after GH-treatment for 3 wk. (C) OGTT and ITT on GH-treated PICK1-deficient mice (black circles) and the saline-treated WT littermate controls (white circles). Significant difference was observed in the OGTT, whereas no difference was observed in ITT. Data are expressed as mean ± SE and analyzed by two-way ANOVA in (A) and Student's <i>t</i> test in (B–D) (<i>n</i> = 4–8 in all the experiments). All experiments have been reproduced in another cohort of mice (<i>n</i> = 3–4), where the age of the mice was 4 wk higher.</p

PICK1 is localized to vesicles exiting the Golgi in cultured GH1 cells, and knockdown of PICK1 reduces GH immunosignal in GH1 cells.

<p>(A) PICK1 is expressed in GH-producing GH1 cells, and PICK1 knockdown reduces GH content. Confocal images of GH1 cells immunostained for PICK1 (blue) and GH (red). Cells transfected with shRNA against PICK1 are identified by obligate coexpression of green fluorescent protein (EmGFP) (green) and are outlined. Insets (squares) show area with (i) no colocalization of PICK1 and GH and (ii) partial colocalization of PICK and GH. Scale bar, 10 µm. (B and C) Quantification of PICK1 immunosignal (B) and GH immunosignal (C) in GFP-positive cells transfected with either of two different shRNAs against PICK1 (52 and 53). Data are % of signal in surrounding non-transfected cells (mean ± SE) for control shRNA (shControl) (<i>n</i> = 48), shPICK1 (52) (<i>n</i> = 69), and shPICK1 (53) (<i>n</i> = 75). ***<i>p</i><0.001, compared to shControl, Mann–Whitney rank sum test. (D) Quantification of the PICK1 co-localization with GH using Van Steensel's cross-correlation function, which reports the Pearson cross-correlation as a function of the relative movement of the two channels with respect to each other. The low but sharp peak close to Δx = 0 indicates partial but specific co-localization. (E) Co-localization of PICK1 with Giantin, TGN38, and syntaxin 6. Confocal laser scanning micrographs of GH1 cells immunostained for endogenous PICK1 (red) and the cis-Golgi marker giantin (top), the trans-Golgi (TGN) marker TGN38 (middle), and the TGN/immature vesicle marker syntaxin 6 (bottom) (all in green). Insets highlight area where the PICK1 signal was either enclosed by, or closely associated with, the Golgi markers (giantin, top, and TGN38, middle) or partially co-localizing (syntaxin 6, bottom). (F) Quantification of PICK1 colocalization with giantin, TGN38, and syntaxin 6 using Van Steensel's cross-correlation function. The broad peak for giantin is characteristic for large adjacent structures, whereas the high and sharp peak close to Δx = 0 for syntaxin 6 indicates specific co-localization. TGN38 is intermediate between these distributions, suggesting a partial overlap of the structure with PICK1. Co-localization was quantified for 10–20 cells from three independent experiments, and data are means ± SE. (G) Dashed box (left) from (E, Bottom) with magnification and gain adjusted for visualization of the punctate staining of PICK1 (red) and syntaxin 6 (green). Intensity profile (right) through several punctae (white line in lower, merged picture on the left) shows peaks corresponding to punctae for both PICK1 and syntaxin 6. All immunocytochemistry data were obtained from at least three independent experiments, and the cells are representative of multiple cells imaged in each experiment.</p

PICK1 and ICA69 co-localize in the Golgi compartment of COS7 cells and promote punctate distribution of GFP-tagged chromogranin A (CgA-GFP) in the cytosol.

<p>(A) Confocal images of COS-7 cells transfected with GFP-PICK1 (top), HA-ICA69 (middle), or GFP-PICK1 and HA-ICA69 (bottom). From left, the channels show GFP-PICK1 in green, HA-ICA69 in red, and endogenous giantin in blue. Upon co-expression, GFP-PICK1 and ICA69 co-localize with giantin in the Golgi compartment. Scale bar, 10 µm. (B) Confocal images of COS-7 cells transfected with CgA-GFP and pcDNA3 (top) or together with mycPICK1 and HA-ICA69 (bottom). From left, the green channel shows CgA-GFP, the red channel shows mycPICK1, and the blue channel shows HA-ICA69. Insets illustrate the marked increase in punctuate CgA structures in cells co-transfected with mycPICK1 and HA-ICA69. Outlines of the cells are shown in the green channel in white. Scale bar, 10 µm. Images are representative of <i>n</i> = 50 (CgA-GFP) and <i>n</i> = 44 (CgA-GFP+mycPICK1+HA-ICA69) cells from three independent sessions. (C) Quantification of the punctuate CgA-GFP signal relative to the total CgA-GFP signal as a measure of the efficiency of Golgi exit. ***<i>p</i><0.001 in Student's <i>t</i> test.</p

Decreased GH storage and secretion in PICK1-deficient mice.

<p>(A) The livers of PICK-1-deficient mice had a significantly lower weight than those of WT mice (**<i>p</i><0.01). (B) PICK1-deficient mice exhibited a decreased level of plasma IGF-1 (*<i>p</i><0.05). (C) Decreased relative expression level of IGF-1 mRNA in the liver of PICK1-deficient mice versus WT mice (**<i>p</i> = 0.0045). Measurements in (A), (B), and (C) were performed in samples from 35–38-wk-old WT (+/+) or PICK1-deficient mice (−/−) after overnight fast. Data are means ± SE (<i>n</i> = 10–14). (D) PICK1-deficient mice display reduced femoral length. Measurements were done on 39–41-wk-old WT (+/+) and PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 4–5). *<i>p</i><0.05, compared to WT. (E) PICK1-deficient mice display decreased ghrelin-induced GH secretion. Basal plasma GH level and the level 5 min after ghrelin administration were determined in anesthetized WT (+/+) and PICK1-deficient mice (−/−) at the age of 7–9 wk. White bars, WT (+/+); black bars, PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 7–9). *<i>p</i><0.05, compared to WT. (F) PICK1-deficient mice display decreased GH content in the pituitary. Pituitaries were taken from 34-wk-old WT (+/+) or PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 4). **<i>p</i><0.01. (G) Relative expression levels of GH and GHR mRNA in pituitaries, determined by RT-PCR analysis (35–38-wk-old mice). White bars, PICK1 +/+: black bars, PICK1 −/−. Data are means ± SE (<i>n</i> = 7–8). *<i>p</i><0.05.</p

Interdependent expression of PICK1 and ICA69 in <i>Drosophila</i>.

<p>(A) Immunostainings for ICA69 and PICK1 in a single peptidergic neuron in the protocerebrum of adult <i>Drosophila</i> brain. HA-tagged ICA69 targeted to peptidergic cells (<i>c929-GAL4/+; UAS-ICA69-HA/+</i>) was detected in parallel with endogenous PICK1. Arrows indicate examples of PICK1/ICA69 co-localization. Scale bar, 10 µm. (B and C) <i>Drosophila</i> ICA69-HA co-immunoprecipitates PICK1 from fly head extracts. Flies were co-expressing ICA69-HA and PICK1 under the pan-neuronal elav-GAL4 driver (<i>elav-GAL4/+; UAS-ICA69-HA/UAS-PICK1A</i>). (B) Immunoprecipitation (IP) with anti-HA antibody and immunoblotting (IB) with anti-PICK1 antibody. Left lane, control without antibody; right lane, lysate. (C) Immunoprecipitation (IP) with rat anti-HA antibody (3F10) and immunoblotting (IB) with mouse anti-HA (16B12) antibody. Left lane, control without antibody; right lane, lysate. (D) Dependence of ICA69 expression on PICK1 expression in <i>Drosophila</i>. ICA69-HA immunoreactivity is shown in control brain (<i>c929-GAL4</i>, <i>PICK¹/+; UAS-ICA69-HA/+</i>) and in PICK1-null mutant brain (<i>c929-GAL4, PICK¹/PICK²; UAS-ICA69-HA/+</i>). pi, pars intercerebralis; pc, protocerebrum; ol, optic lobe. Pictures are representative of six fly brains stained in three experiments. (E) Dependence of PICK1 expression on ICA69 expression in <i>Drosophila</i>. PICK1 immunosignal in control brain without RNAi transgene (<i>ELAV-GAL4/UAS-DCR2;;TM3/+</i>) and in brain with pan-neuronal expression of ICA69 hairpin RNA (<i>ELAV-GAL4/UAS-DCR2;;UAS-ICA-RNAi/+</i>) (ICA69-RNAi). Scale bar, 100 µm. Pictures are representative of six fly brains stained in three experiments. (F) Western blotting of head extracts from control and ICA69 RNAi flies. (G) Quantification of the data in (F). Protein levels are normalized to ELAV. Data are means ± SE, <i>n</i> = 6, <i>p</i><0.05. (H and I) Quantification by RT-PCR of ICA69 mRNA (H) and PICK1 mRNA (I) levels in heads from <i>ICA69</i> knockdown (ICA69-RNAi), <i>PICK1¹</i>, and control flies. Data are means ± SE, <i>n</i> = 6 for control and ICA69-RNAi, <i>n</i> = 4 for <i>PICK1¹</i>, *<i>p</i><0.05, **<i>p</i><0.01.</p