11 Flashcards
Presynaptic regulation of neurotransmitter levels
• Ca2+ influx leads to activation of protein kinases (e.g., PKA, CaMKII, MAPK)
• Tyrosine hydroxylase is phosphorylated, leading to increased catecholamine synthesis
• Increased neurotransmitter release into synaptic cleft
• Longer-term changes in tyrosine hydroxylase mRNA expression can be modulated by hormones (e.g.
glucocorticoids)
Nerve Growth Factor (NGF)
- Neuropeptide
- Promotes neuron survival
- Dysregulation of NGF signaling has been implicated in neurodegenerative disorders
- Increasing NGF levels in basal forebrain (e.g., viral therapy) may ameliorate neurodegeneration
Brain-derived neurotrophic factor (BDNF)
- Neuropeptide
- Promotes neuron survival
- Promotes growth of dendrites and synapses
- Involved in learning and memory later in mature animals
- Enhanced by environmental enrichment
- Dysregulation of BDNF signaling (e.g., val66met mutation) common in patients suffering from neurodegenerative and other neurological disorders (e.g., anxiety disorders)
- Thus far, treatments that increase BDNF levels in the brain have not been successful
Steroid hormones
steroid hormone + protein carrier cross cell membrane binds to receptor protein becomes transcription factor mRNA read by ribosomes protein secreted
Olfaction
is an important sense for many mammals
Humans are an exception
Olfactory receptors
• Olfactory receptor neurons are embedded in the nasal epithelium
• The olfactory receptor neurons have cilia that extend into
the nasal cavity, where they are exposed to inhaled odorants
• The olfactory receptor neuron cilia are covered in odorant receptors, which are all 7-transmembrane domain GPCRs
• Each olfactory receptor neuron only expresses one type of olfactory receptor
Olfactory receptors structure
- Highly conserved 7TM structure
- Many genes (~1000 in mammals) express different receptors (~3% of the genes in genome!)
- Only receptor in each olfactory neuron
The combinatorial capacity of olfactory receptors leads to a large number of differentiable odors.
Similar molecules can smell very different
Short-term effects
Direct effects on membrane potential are generally rapid, but short-lasting
Channels can also be opened by second messengers
Intermediate-term effects (kinases and phosphatases)
Activation of kinases is fairly rapid, but can have longer
lasting effects than direct effects on membrane potential
Ca2+ > CaMKII cAMP > PKA
cGMP > PKG DAG > PKC
Why is phosphorylation used in intracellular signal cascades?
Phosphorylation is the most abundant post-transcriptional
modification (~50% of eukaryotic proteins switch between
phosphorylated and dephosphorylated states)
Phosphorylation changes the tertiary structure of the protein, leading to a conformational change which can either activate or inactivate the target protein
CEBPB (transcription factor), activated by cAMP/PKA
What is a protein kinase?
Protein kinases transfer the phosphate group from ATP to a particular amino acid residue on the target protein (many
kinases are quite specific for a small number of proteins)
Only certain residues can be phosphorylated: Serine (Ser),
Threonine (Thr), and Tyrosine (Tyr). The residue also must be exposed (most proteins have particular phosphorylation “sites”)
Protein kinases are usually specific for which residue they
phosphorylate: PKA, PKC, CaMKII modify Ser/Thr; while
receptor tyrosine kinases modify Tyr
Protein kinase A (cAMP)
PKA is a tetramer, with two regulatory subunits and two catalytic subunits. When inactive the regulatory subunits prevent the catalytic subunits from phosphorylating proteins
Binding of cAMP to PKA causes catalytic subunits to dissociate, allowing them to phosphorylate numerous proteins (amplification)
Protein kinase C (PLC/DAG) structure
PKC is a monomer with an autoinhibitory domain
DAG/Ca2+ binding recruits PKC to the plasma membrane and releases autoinhibition of the catalytic domain
The unbound catalytic domain phosphorylates target proteins
Protein kinase C (PLC/DAG) mechanism
(1) Ca2+ influx induces binding of PKC to membrane
(2) DAG removes first regulatory domain
(3) Additional DAG removes second regulatory domain and frees catalytic domain
CaMKII (Ca2+/calmodulin) structure
CaMKII is a monomer with an autoinhibitory domain
CaMKII can itself be phosphorylated (called autophosphorylation), leading to persistent activity.
Autophosphorylation is thought to be very important for synaptic plasticity
Calmodulin structure
Calmodulin has four Ca2+ binding sites
Ca2+ binding induces conformational change, exposes regions (red asterisks) that bind to target proteins
Crucial for activation of CaMKII, but also plays other functions
CaMKII (Ca2+/calmodulin-dependent protein kinase II)
CaMKII assembles into multimers of 8-14 subunits (a and b types in the brain), each with an independent kinase domain
CaMKII accounts for 1-2% of all protein in the forebrain
mutating CaMKII
Mutating the CaMKII Thr286 residue (phosphorylation site)
to an Alanine prevents autophosphorylation
This mutation resulted in profound deficits in synaptic
plasticity and learning and memory
What is a protein phosphatase?
Protein phosphatases remove the phosphate group from a
phosphorylated protein (does not turn back into ATP)
They are generally less specific for particular proteins than kinases
Example phosphatases include PP1-6 (Ser/Thr), PTEN (Tyr), and DUSP1-29 (dual-specificity). PP2B (also known as calcineurin) is unique in that it is activated by Ca2+/calmodulin
Phosphatases are crucial for proper synaptic function
Intermediate-term effects (receptor trafficking)
Activation of kinases/phosphatases can result in trafficking of receptors into or out of the synapse
Second messenger targets can be diverse
The same GPCR (mAChR) can have both direct effects (opening K+ channel) and (bidirectional) downstream effects via second messengers(phosphorylation/dephosphorylation)
long-term effects (transcriptional modification)
Transcription (DNA > RNA)
(1) RNA polymerase assembles on promoter region of DNA
(2) RNA polymerase separates the two DNA strands and makes a single-stranded RNA copy (messenger RNA or mRNA)
Translation (mRNA > Protein)
(1) Ribosome (green) assembles around mRNA, tRNA binds to Start codon
(2) Ribosome translocates down RNA; tRNAs bind to each 3-nucleotide codon, each transferring an amino acid to the growing polypeptide
(3) When stop codon is reached, ribosome cleaves polypeptide
(4) The polypeptide will later fold into the functional protein
where does Transcription and Translation occur
Transcription is always in the nucleus
Translation is in the ER or cytoplasm (including processes)
Second Messenger Targets:
- Second messengers are now thought to affect translation (e.g., by phosphorylation of eukaryotic initiation factor (EIF) proteins)
- Transcriptional modification: Several kinases (PKA, CaMKII, MAP kinase) have been shown to phosphorylate transcriptional activators, upregulating transcription of particular RNA transcripts
- The changes are slow-acting (30-60 minutes) and can have long-lasting effects (hours to days)
what is CREB
Transcriptional activator proteins (e.g., CREB) regulate RNA
polymerase binding
The probability of transcription can vary depending on whether the transcriptional activator protein is phosphorylated
structure of CREB
A ubiquitous transcriptional activator protein that can be
phosphorylated by PKA, CaMKII or MAPK - leading to increased transcription of: c-fos, arc (IEGs), and BDNF mRNAs, among others
CREB plays a crucial role in:
- Cell Survival (CREB knock outs tend to be lethal)
- Learning and Memory (knockdowns result in memory impairments)
- Circadian rhythms (knockdowns result in sleep disord
CREB is also thought to play a role in several disease phenotypes
Immediate Early Genes (IEGs)
are genes that are activated rapidly (~30-90 minutes) and transiently in response to strong cellular stimuli
• Examples include c-fos, c-myc, arc, and zif268
• The proteins from these genes can act as transcriptional
activators for a variety of additional genes (called delayed
response genes)
• Many of these genes are crucial for learning and memory,
and are often used as markers for neurons that have
undergone synaptic modifications
Using IEGs to “tag” cells involved in a memory
cFos-tagged cells express channelrhodopsin, allowing them to be reactivated at a later time
Context memories can be artificially evoked by
activation of “cFos-tagged” cells
Insertion of AMPA receptors
- NMDA receptor opening leads to Ca2+ influx
- Increased Ca2+ leads to activation of CaMKII and PKC
- CaMKII and PKC phosphorylate additional proteins (SAP-97 and Myosin-VI, etc.), increasing trafficking of AMPA receptors to membrane (perisynaptic)
- CaMKII can also activate CREB, which increases transcription of AMPA receptor mRNA
Internalization of AMPA receptors
• Ca2+ influx leads to phosphatase activation (e.g., PP1, calcineurin).
• Phosphatases dephosphorylate proteins involved in insertion (SAP97 and Myosin-VI, etc.). Calcineurin activates dynamin
• AMPA receptors are internalized into cytoplasmic vesicles, where they can be recycled or targeted to
the lysosome for degradation
