mf_ 6_receptorsI - Receptors and Signaling I: General...

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Unformatted text preview: Receptors and Signaling I: General Features of Receptors Receptors are specific Receptors are sensitive high affinity amplification adaptation Different cells have different Different receptors receptors Receptors can be Receptors intracellular or at the cell surface surface Cell surface - growth factor Cell Intracellular- steroid hormone receptor receptor Physical/Chemical Signals Changes Sensed by Receptors Physical Cell binds to other cell Cell Binds to Cell extracellular matrix extracellular Chemical Acetylcholine Peptide Hormone Growth Factor Cholesterol Metabolism Immune response Nervous system Sensory Transduction Cells that Cells produce/receive signal may be near or far far Receptor Functions Function of Cell Surface Receptors Adhesion Acquire Material for Cell LDL Transferrin Receive Information Pass Information to Cell Amplify Signal Amplify Few molecules of signal and Few receptor receptor Amplification Second Messengers Enzyme Cascades Utilize Energy Phosphorylation G protein protein Signal Integration Adaptation Modulate sensitivity to level of Modulate signal signal Adaptation The same hormone produces different responses in different cells: Acetylcholine Muscarinic Ach Muscarinic Rec Rec Heart Salivary Gland Salivary Nicotinic Ach Rec Skeletal Muscle Mechanisms of Adaptation Modulate response Modulate Decrease sensitivity Too much response, or too little response can be harmful Cancer Allergy Diabetes Ligand Binding Receptors Characterization of Receptors and Pathways Ligand Binding Number of Receptors Affinity of Receptors Scatchard Plot for Analysis Specific vs Non-Specific Specific Binding Binding How do they measure specific How binding? binding? Inhibitors Affinity Chromatography Molecular Genetics Characterize Receptor Characterize Trafficking Trafficking Internalization Degradation Characterize Signaling Pathway Genetics Biochemistry Microscopy 3 Modes Signaling Three Primary Modes of Receptor Action Neuromuscular Junction Neuromuscular Acetylcholine Receptor Acetylcholine Receptor Structure Acetylcholine 2 types of Acetylcholine Receptors (See Fig. 15-9) (See Muscarinic 7 TM spans G protein Nicotinic Ion channel Neuromuscular Junction Neuromuscular Subunits: α 2 , β, γ , δ α subunit binds ligand Both α must bind to open Structure Conserved from Mammal to Electric Eel Structure From Study of Eel Receptors Biochemistry Molecular Genetics Electron Microscopy 3 conformations Three Conformations of the Acetylcholine Recepto Three Channel Closed Ligand Binds Channel Opens Na+ influx Channel Inactivates Analogous to 3 states of voltage gated Na+ channel Molecular Genetics Probing the Structure of the Acetylcholine Receptor Using Molecular Genetics Transcribe mRNAs for bovine Ach R α, β, γ , δ Transcribe Inject Xenopus oocytes (frog eggs), and study channels with Inject patch clamp patch Normal frog eggs do not have any acetylcholine receptors Repeat for Torpedo eel Ach R α, β, γ , δ Repeat See difference in cow and eel receptor by patch clamp Try to understand which subunits confer differences by mixing Mix mRNAs to make hybrid channels: results show δ is important Mix hybrid is Make chimeric subunits by splicing DNA to identify part of δ subunit that is critical: focus on second transmembrane region M2 that Point mutations to find critical region in M2 that forms pore Hybrid Channels Structure/Function Studies of Ach Receptors: Hybrid Channels Channels B. Sakmann. Science 256, 503 - 513 (1992) Xenopus Expression System Inject mRNA for α, β, γ , δ Inject Hybrid Channels: Mix Subunits from Different Sources Bovine Bovine (msec) (msec) Torpedo t1/2 at 100 mV 7.6 0.6 α, β, γ , δ α , β, γ , δ δ α, β, γ 8.6 If only delta was from cow and other subunits from Torpedo eel, then the hybrid channel remained open like a cow channel. Decided to focus on the delta subunit. Chimeric Channels Structure/Function Studies of Ach Receptors: Chimeric Channels Channels B. Sakmann. Science 256, 503 - 513 (1992) Chimeric Channels: Splice cDNA for one subunit from Different Sources Xenopus Expression System Xenopus Inject mRNA for α , β, γ from Inject eel, and chimeric δ subunit. The delta subunit behaves like cow or eel based only on whether the M2 part of delta is from cow or eel . Later, they made single amino acid substitutions in this region to identify the amino acids that form the narrowest part of the pore opening. Proposed M2 segments of all Electron Microscopy Gating Mechanism of Ach Channel Revealed by Electron Microscopy Revealed Lodish 5th 7-46 Acetylcholine Receptor Electron Microscopy Nigel Unwin Eel membranes Closed Structure Open Structure of Channel spritz Ach &quick freeze Compare open and closed Double Leucine ring is gate All 5 Helices rotate to open Also See Fig. 11-38b ACH Receptor Summary Acetylcholine Channel Structure Revealed by Chimeric Channels, Point Mutations and Electron Microscopy M2 transmembrane regions of all subunits have M2 all conserved features conserved M2 is critical for the conductance of the pore M2 The M2 segment from each of the 5 subunits lines the pore Hydrophobic Leucine Leucine is the Gate 5 subunits with 2 leucines in each 10 leucines block pore in closed state Rotation of M2 helices away from the center Rotation opens pore opens Shown by electron microscopy of channel frozen in open Shown state state Binding of Ach induces conformational change to open Binding channel. channel. Channel Opening Occurs by Rotation of Transmembrane Helices Lining the Pore ...
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