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Outline
• From extracellular signal to cellular response • Cell surface receptors and signal transduction proteins • G-protein coupled receptors (GPCRs): structure and mechanism • GPCRs that regulate ion channels • GPCRs that activate or inhibit adenylyl cyclase • GPCRs that trigger elevation in cytosolic Ca2+
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6. Study the receptors and activity of signal transduction proteins
1) Purification of receptors
• • Recombinant DNA Affinity chromatography
2) Study the activity of signal transduction protein
＜108 mol/L
high affinity
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1) Eight classes of cell-surface receptors
Signaling via Enzyme -Linked Cell -Surface Receptor
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Molecular Cell Biology，6th ed., H. Lodish, 2007
cellular responses
• This chain of intermediates: a signal transduction pathway.
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1. Signaling molecules
1) Intercellular & intracellular signaling molecules
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2) Signaling molecules act locally or at a distance
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3) NO and CO
The Nobel Prize in Physiology or Medicine 1998
"for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system"
Robert F. Furchgott 1/3 of the prize USA 1916 ~
Louis J. Ignarro 1/3 of the prize USA 1941~
Ferid Murad 1/3 of the prize USA 1936~
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• Robert F. Furchgott’s work EDRF (endothelium-derived relaxing factor)
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2) Types of protein kinases and phosphatases
• About 600 protein kinases and 100 phosphatases • Two types of protein kinases: add phosphate to the hydroxyl group on tyrosine residues add phosphate to the hydroxyl group on sersine/threonine residues • The receptor possesses intrinsic kinase activity or tightly bound to a kinase.
•
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Four common intracellular second messengers
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2) Advantages of second messengers
• Diffuse through the cytosol much faster than proteins • Facilitate amplification of extracellular signal
Pathways involving proteolysis
Molecular Cell Biology，6th ed., H. Lodish, 2007
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2) Signaling molecules binding to intracellular receptors
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3) TheΒιβλιοθήκη steroid receptor superfamily
• • Immunoprecipitation of kinases Pull down assays of GTP-binding proteins
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Activation of three signal transduction proteins by phosphorylation
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A pull-down assay shows that the small GTPbinding protein Rac1 is
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6) Maximal cellular response usually does not require activation of all receptors
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7) Sensitivity of a cell to external signals
1) The fewer receptors present on a cell, the less sensitive the cell is to the ligand. 2) Hypersensitivity (such as HER2)
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3) Measure the affinity of a receptor for its ligand a) Dissociation constant
• • Kd=[R][L]/[RL] Kd=[L] which half of the receptors have a ligand bound when the system at equilibrium
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b) Binding assays to determine the Kd
• For receptors have high affinity for their ligand
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• For low-affinity ligands
Low-affinity
Low-affinity
High-affinity
3) Desensitization
regulation at the levels of transcription, translation and posttranslational processing, controlling the rate of degradation.
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3. Cell-surface receptors & intracellular receptors
1) High affinity:
Receptor binds only a single signaling molecule or a group of structurally very closely related molecules.
2) High specificity:
The ability of receptors bind or not bind closely related substances.
48 nuclear receptors
(human), 2 categories: classical endocrine hormones receptors and orphan receptors.
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4. Protein kinases and phosphatases
1) Phosphorylation and dephosphorylation: common cellular mechanism for regulating protein activity
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A simple signal transduction pathway involving one kinase and one target protein
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5. Intracellular second messengers
1) Second messengers
• The binding of ligands (first messengers) to many cellsurface receptors leads to a short-lived increase (or decrease) in the concentration of certain low-molecular-weight intracellular signaling molecules termed second messengers. Such as Ca2+ , cAMP, cGMP, DAG, IP3
Signal Transduction and G Protein Coupled Receptors
Han Yingying Dept. of Biochemistry and Molecular Biology
Tel:69156428 E-mail:151259808@ Sep.19 2014
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4) Molecular complementary
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