Pichlo M et al. (2014), High density and ligand affinity confer ultrase...

ECB-ART-43573

J Cell Biol 2014 Aug 18;2064:541-57. doi: 10.1083/jcb.201402027.

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High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

Pichlo M , Bungert-Plümke S , Weyand I , Seifert R , Bönigk W , Strünker T , Kashikar ND , Goodwin N , Müller A , Pelzer P , Van Q , Enderlein J , Klemm C , Krause E , Trötschel C , Poetsch A , Kremmer E , Kaupp UB , Körschen HG , Collienne U .

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Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3'',5''-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons.

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Genes referenced: LOC100887844 LOC100893907 LOC115919910 LOC576733 LOC583082 LOC594261 ngly1

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	Figure 1. Ligand affinity of sperm GC. (A) Dose–response relation of resact binding. The continuous line represents a fit of the Hill equation to the data from a total of 126 experiments. Each data point represents the mean ± SD of at least three experiments. The constant of half-maximal binding was K1/2 = 0.65 ± 0.08 nM, and the Hill coefficient was n = 0.49 ± 0.03. The occupancy at ∼750 nM resact was set to 95% (31 experiments; red dot). (B) Dose–response relation at low resact concentrations. The solid line was calculated using a simple binding model using a Kd of 250 pM.
	Figure 2. Turnover number and inactivation kinetics of GC. (A) Time course of cGMP production in the presence of 1 mM IBMX at different resact concentrations. Error bars are means ± SD from at least three experiments. (B) Time course of cGMP synthesis (red triangles) for sperm stimulated with 250 nM resact. The apparent turnover number of GC (black squares) was calculated by using Eqs. 1–3 (Materials and methods). The data shown are from a single representative experiment out of 12 from six different animals. Lines represent exponential fits. (C and D) Turnover number (C) and inactivation rate of GC (D) versus resact concentration. Each data point represents the mean ± SD. Dashed lines denote the mean turnover number and inactivation rate over all concentrations, respectively (T = 72.2 ± 28.7 cGMP molecules/second; ki = 6.4 ± 2.4 s−1). Number of experiments is given in parentheses above the data points. Turnover numbers and inactivation rates were calculated from time courses of cGMP.
	Figure 3. Kinetics of dephosphorylation. (A) Representative Western blot of GC dephosphorylation at different resact concentrations. The amount of GC was adjusted to 120 nM. Sperm were stimulated with 2.5, 25, 250, or 500 nM resact. The time lapse in seconds after resact application is indicated above the respective lanes. The molecular mass of a protein size marker is shown on the right. (B) Fraction of dephosphorylated and phosphorylated GC versus time after stimulation with 250 nM resact. Inset shows the dephosphorylation reaction during the first 200 ms. Data shown are from a single representative experiment out of 12 from six different animals. Lines represent exponential fits. (C) Time course of dephosphorylation for resact concentrations from 50 to 500 nM. The time constant (τdephos = 133 ms) was obtained by using a global fit that includes ligand binding and unbinding. Each data point represents the mean ± SD of at least three experiments. The inset shows the dephosphorylation reaction during the first 2 s. (D) Comparison of the time course of relative turnover number and dephosphorylation. Both time courses were normalized according to the minimum–maximum method. The data shown are from a single representative experiment out of 12 from six different animals. (E) Amount of GCdephos after 3 min of incubation with different resact concentrations. Total GC concentration was adjusted to 120 nM. Each data point represents the mean ± SD. Number of experiments is given in parentheses. Lines represent exponential fits in B, D, and E.
	Figure 4. GC is inactivated by dephosphorylation. (A and B) Western blots showing dephosphorylation of GC induced by intracellular alkalization. pHi was elevated by different concentrations of NH4Cl (A) or TMA (B). The concentrations of the base and incubation times (minutes) are indicated above the respective lanes. The molecular masses of two protein size markers are shown on the left. (C) Representative Western blot of dephosphorylation by alkaline pH. (right) First, GC was dephosphorylated by incubation of sperm with 122 mM TMA for 15 min. Subsequently, sperm were stimulated with 250 nM resact. The time lapse after resact stimulation (seconds) is indicated above the respective lines. The molecular masses of two protein size markers are shown on the left. The left shows control dephosphorylation after resact stimulation in the absence of TMA. (D) Time course of cGMP synthesis in the presence of 2 mM IBMX after alkaline dephosphorylation. Control without TMA preincubation is shown. Lines represent exponential fits. Each data point represents the mean ± SD of three experiments based on duplicates.
	Figure 5. The recombinant GC is catalytically inactive. (A) Immunocytochemical staining of cells (top) or membrane sheets (bottom) of a cell line expressing a cGMP sensor (green) and GC (red). Bars, 20 µm. (B) Fluorescence images taken during a Ca2+ imaging experiment. Transfected HEK293 cells were loaded with Fluo-4-AM and incubated with 200 µM IBMX for 10 min. Cells were stimulated with 200 µM resact and subsequently with 3 mM 8-Br-cGMP. The increase of [Ca2+]i is shown in false colors. Bars, 50 µm. (C) Mean change in fluorescence (ΔF) for the experiment (19 cells) shown in B. The addition of 200 µM resact and 3 mM 8-Br-cGMP is indicated by arrows. (D) Membrane proteins of sea urchin sperm (Sp) after resact stimulation and of the double-stable cell line were analyzed by “dirty” SDS-PAGE (−PNGase). To compare the phosphorylation state of the native and recombinant GC, both were deglycosylated by PNGase. After deglycosylation (+PNGase), the molecular mass of recombinant GC and dephosphorylated GC from sea urchin sperm are almost identical. The molecular masses of two protein size markers are shown on the right.
	Figure 6. Dephosphorylation is independent of signaling events downstream of GC. (A–F) Signaling events after stimulation with 25 nM resact at different extracellular K+ concentrations ([K+]ex). Each trace represents the mean of at least three recordings. (A) Changes in Vm detected by di-8-ANEPPS. The dashed line indicates the resting potential. (B) Changes in pHi detected by BCECF. ΔR represents the ratio of fluorescence at 540 and 494 nm; excitation is at 425 nm. (C) Changes in [Ca2+]i detected by Fluo-4. (D) Percentage of dephosphorylated GC after 3 min of stimulation with 25 nM resact at different [K+]ex. (E) Apparent time constant τ of dephosphorylation at [K+]ex. (F) Changes in cGMP concentration after resact stimulation at 9 and 58 mM [K+]ex in the presence of 2 mM IBMX. Lines represent exponential fits. D–F represent the means ± SD of six experiments based on duplicates. (G) Resact-induced dephosphorylation in ASW or with additional 1 µM calyculin A or 1 µM okadaic acid. (H) Resact-induced dephosphorylation in ASW or with additional 10 µM sanguinarine chloride. In G and H, the amount of GC was adjusted to 120 nM, and sperm were stimulated with 250 nM resact in the presence or absence of the respective phosphatase inhibitors. Time after resact stimulation (seconds) is indicated above the respective lanes. The molecular masses of two protein size markers are shown on the left.
	Figure 7. Limiting accuracy of molecule detection. (A) Uncertainty δc/c for the detection of resact versus the receptor number NR for different resact concentrations. Because the effective radius s of the binding site is not known, the radii of the globular extracellular domain and of the ligand were used as an upper and lower estimate, respectively. (B) The resact structure was modeled using PEP-FOLD (Maupetit et al., 2009, 2010; Thévenet et al., 2012) with a disulfide bridge between C1 and C8. The model was visualized using RasTop, a tool based on RasMol 2.7. (C) Schematic distribution of a GC and CNGK on the flagellum. The scheme depicts the intracellular site of the flagellar membrane. An active GC dimer is depicted in red, an inactive GC dimer is shown in gray, CNGK is depicted in green, and the gradient of cGMP concentration is given in shades of magenta. The GC and CNGK densities are shown to scale. The GC dimer/CNGK ratio is 10:1.

References [+] :

Aburai, Sanguinarine as a potent and specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via phosphorylation of p38 in HL60 cells. 2010, Pubmed