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Cellular NeurobiologyBIPN140
1st Midterm Exam Ready for PickupBy the elevator on the 3rd Floor of Pacific Hall (waiver)
Exam Depot Window at the north entrance to Pacific Hall (no waiver)Mon-Fri, 10:00 AM to 4:00 PM
1st midterm regrade: contact the IA who graded the question directly before Nov 14, 2016.
PS4 Q&A will be posted on October 27th
Nick’s Office Hour this Wednesday (Oct 26) is canceled.
Chih-Ying’s Office Hour: Monday, 1:00-2:00 PM, Bonner Hall 4146
BIPN140 Lecture 10: Intracellular Signaling
Su (FA16)
1. Overview of Chemical Signaling
2. G protein signaling & Phosphorylation
3. Nuclear Signaling
4. Receptor Tyrosine Kinase Signaling
Chemical Signaling in Neurons (Fig. 7.1)
Amplification in Signal Transduction Pathways (Fig. 7.2)
Enzyme Enzyme
Depends on the half life of the activated receptor
Depends on the half life of the GTP-bound G
Three Classes of Cell Signaling Molecules (Fig. 7.3)
Lipophilic (e.g. steroidhormone)
Categories of Cellular Receptors for Secreted Signals (Fig. 7.4)
e.g. membrane bound guanylyl cyclase
Types of GTP-binding Proteins: Large G Proteins (Fig. 7.5)
GEFs: Guanine nucleotide exchange factors
GAPs: GTPase activating proteins (the endogenous GTPase activity is usually too slow to terminate the signal)
Effector Pathways Associated with GPCRs (Fig. 7.6)
Types of GTP-binding Proteins: Small G Proteins (Fig. 7.5)
GEFs: Guanine nucleotide exchange factors
GAPs: GTPase activating proteins (the endogenous GTPase activity is usually too slow to terminate the signal)
Neuronal Second Messengers (Fig. 7.7B-D)
Ca2+-induced Ca2+ release (CICR)
Membrane phospholipid
Neuronal Second Messengers (Fig. 7.7A)
Gs
Gi/o
Gq
Extracellular Ca2+
Intracellular Ca2+
Different PLC isoforms are differentially regulated by Gq, Ca2+, and receptor tyrosine kinase
Requires ATP
Powered by Na+ gradient
Regulation of Cellular Proteins by Phosphorylation (Fig. 7.8)
Phosphorylation Sites
(Mendez et al., Neuron 28, 153-164, 2000)
Mechanism of Protein Kinase Activation (Fig. 7.9)(cAMP-dependent protein kinase)
(calcium/calmodulin-dependent protein kinase II )
(protein kinase C)
Regulation of Tyrosine Hydroxylase by Phosphorylation (Fig. 7.14)
Receptor Tyrosine Kinases
Kandel et al., Principles of Neural Science, 5th Edition, Figure 11-9
Ligand bindingDimerizationPhosphorylation of its partner
Receptor auto-phosphorylationRecruitment of adaptor proteinsDifferent downstream signaling events
Mitogen-activated protein kinase (MAPK): activate other kinases by direct phosphorylation (MAPKK or MAP2K, MAP3K, MAP4K)
GRB2
GEF
Tyrosine Kinases (Fig. 7.12)
Nuclear Signaling/Transcription (Fig. 7.10)
Upstream activator site
Transcriptional Regulation by CREB (Fig. 7.11)
Background: Target tissues of sympathetic and sensory neurons secret NGF to promote nerve growth/differentiation and survival. The neurotrophic effect of NGF requires activation of CREB transcription factor at the cell body. However, it is unclear how NGF signaling at the axon terminal is propagated back to the soma which can be over 1 meter away. Is NGF internalized by itself with activated TrkA back to the cell body for the transmission of the signal? Alternatively, is some downstream signaling proteins (ras, MAPK?) or 2nd
messengers generated at the axon terminal upon NGF-TrkA interaction transported back to the cell body?
Experiments: Use compartmentalized cultures of sympathetic neurons (axon terminals and cell bodies are cultured in different chambers). Generate phospho-specific antibodies that label site specific-phosphorylated and unphosphorylated CREB (anti-P-CREB) and TrkA (anti-P-TrkA). This system enables exposure of terminals or cell bodies to NGF and then to assess by immunocytochemistry the phosphorylation (activation) state of CREB and TrkAin cell bodies.
1. Maintain the culture in low NGF (2 ng/ml).2. Stimulate the culture with high NGF (200 ng/ml) at
either axon terminals or cell bodies.3. Immuno-stain with anti-P-CREB and/ot anti-P-TrkA4. Speed: 2-4 mm/hour
Whole-cell lysates (controls)
Cell bodies
Axon terminals and distal processes
Cell bodies
Axon terminals and distal processes (center-plated)
Side-plated
Fig. 1. Phosphorylation of CREB after application of NGF to sympathetic neurons
Campenot Chamber
anti-P-CREB
Fig. 2. Internalization of NGF is required for CREB activation at cell bodies
NGP-coupled microbeads: can activate TrkA but can not be internalized
anti-P-TrkA
Results: Internalization of NGF and its receptor TrkA, and their transport to the cell body, were required for transmission of the transmission of target-derived NGF signaling. The tyrosine kinase activity of TrkA was required to maintain it in an auto-phosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus the NGF-TrkAcomplex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.