(2) signal transduction mechanisms

neurophysiology 2: cellular signal transduction mechanisms, ionotropic receptors, g protein coupled receptors (GPCRs), tyrosine kinase linked & nuclear receptors

what are ionotropic receptors also known as, how are they controlled?

• aka ligand gated ion channel

• they are integral membrane proteins that open an ion conduction pathway whne activated by a ligand signal/chem ligand binding

• somewhat ion-selective

what are 4 major ionotropic receptors and their plasma membrane?

1. nicotinic ACh receptor (Cholinergic)

2. AMPA receptor (Glutamatergic)

3. NMDA receptor (Glutamatergic)

4. GABA receptor (GABAergic)

what is cholinergic ionotropic receptor? (what receptor does it use, what neurotransmitter/activator it use, and what conducting ions)

• uses nicotinic acetylcholine receptor

• activated by acetylcholine/nicotine

• lets inward flow of Na⁺ and Ca²⁺ across plasma membrane

• EXCITATORY (+ in)

what is glutamatergic ionotropic receptor? (what receptor does it use, what neurotransmitter/activator it use, and what conducting ions)

• use AMPA or NMDA receptor

• activated by glutamate and/or glycine (both for NMDA)

• inward flow of Na⁺ and Ca²⁺ across plasma membrane

• EXCITATORY

what is GABAergic ionotropic receptor? (what receptor does it use, what neurotransmitter/activator it use, and what conducting ions)

• uses GABA receptor

• activated by gamma-aminobutyric acid (GABA)

• inward flow of Cl^- across plasma membrane

• INHIBITORY (let - in)

what are nicotinic acetylcholine receptors (nAChRs) and how do they work?

• pentamer

• acetylcholine or nicotine (similar shape as ACh) binds to receptor, OPENING conduction pathway to let major Na⁺ flow INSIDE

• result in FAST depolarization/EXCITATORY signals

how do muscarinic acetylcholine receptors (mAChRs) work

• acetylcholine binds to receptor

• G-protein coupled receptor (GPCR) mediate enzymatic reaction, causing intracellular chemical signal cascade with downstream effector target

• SLOWer response than nAChRs

what are the two general receptor themes of glutamate receptors? give examples

1. ionotropic : AMPA, NMDA (learning & memory; neuron excitotoxicity; depression)

2. metabotropic: mGLuR GPCRs

describe AMPA glutamate receptor (description, requirements for activation, how it activates, what pathway it opens)

• tetramer

• need at least 2 ligand mc (usually glutamate NT) for activation

• binding changes shape in ligand binding, leads to shape change in transmembrane, leads to activation

• opens clear Na⁺ conduction pathway

name and locate the receptor domains: NTD, LBD, TMD, CTD

NTD: N-terminal (top one)

LBD: ligand-binding (second one next to Glu)

TMD: transmembrane (near yellow membrane)

CTD: C-terminal (bottom)

describe NMDA glutamate receptor (description, requirements for activation, how it , what pathway it opens)

• tetramer

• requires binding of glutamate AND glycine for activation

• Mg²⁺ will sit in pore to BLOCK conduction pathway at -70 mV

• Mg²⁺ is pushed OUT once cell depolarizes due to AMPARs

• opens the Ca²⁺ conduction pathway

what are the two general receptor themes of GABA receptors

• ionotropic: GABA_A receptors

• metabotropic: GABA_B receptors

what are GABA_A receptors are how do they work (binding, what pathway, what flow)

• pentamer like nAChR

• GABA binding activates receptor

• opens Cl^- conduction pathway

• inward Cl^- flow promotes INHIBITION (hyperpol) and make potential MORE NEG

where are acetylcholine receptors found

• cholinergic, nicotinic, and muscarinic synapses

• synapses have the respective receptors that receive ACh from the neurons

• acetylcholinesterase is near receptor to break down ACh, stopping signal

what are the 4 steps of ionotropic receptor at the post synaptic neuron

1. action potential arrives in presynaptic signal, causing

2. neuron to RELEASE NeuroTransmitters across synapse

3. NTs reach and bind postsynaptic ionotropic receptor, inducing POSITIVE ion conducting pathway (Na, Ca) - EXCITATORY

4. POSITIVE charge diffuses down more + membrane, activating other voltage-gated ion channels to create another AP and so on…

how many times does the serpentine receptor go in and out of the cell membrane

7 times - aka 7-transmembrane (7TM) receptor

what are the 2 GPCR-based drugs focused on in class? name the drug, the GPCR target and disease

1. drug: Zyprexa ; GPCR: serotonin receptor ; disease: schizophrenia

2. drug: Plavix ; GPCR: ADP receptor ; disease: stroke

what accounts for the diversity of metabotropic responses and why

GPCR because it has many active sites

what is a G protein? describe its structure and how its activated/inactivated

• heterotrimeric protein

• subunits are α, β, γ

• alpha subunit is activated by GTP binding, inactivated by GDP

• not as diverse as receptor

what is a guanine nucleotide exchange factor (GEF) and what does it do

• separate regulatory protein

• attaches to G proteins to promote GDP dissociation and GTP binding

• turns signals ON

• for G_a are activated cell surface receptors

what is a GTPase activating protein (GAP) and what does it do

• separate regulatory protein

• promotes hydrolysis of GTP to GDP by binding to G protein

• turns signal OFF

• intrinsic to the G_a protein itself (Ga protein performs it itself)

how is protein kinase A activated in the cAMP pathway (6)? name the 1st and 2nd msngr

1. neurotransmitter(1st msngr) binds to specific receptor (GPCR)

2. occupied receptor cause replacement (GEF) of GDP bound to Gs by GTP, activating Gs

3. Gs (alpha subunit) activates adenylyl cyclase

4. adenylyl cyclase catalyze formation of ATP to cAMP (2nd msngr)

5. cAMP activates PKA

6. PKA phosphorylate other proteins and ion channels

go through the calcium/IP3 pathway (Gq) , underline the two 2nd msngrs and where they go

1. signal binds to 7TM G-protein linked receptor on cell outside

2. Gq a-subunit is activated by swapping GDP for GTP (GEF func)

3. Gq a-subunit activates PLC, which CUTS PIP2 into

   1. IP3: leaves membrane and floats into cytoplasm twd ER

   2. DAG: stays inside cell membrane

4. ER releases Ca2+ from IP3-gated Ca release channel

5. Protein kinase C is activated by released Ca2+ and DAG

what specific trigger causes the store-operated Ca²⁺ channels (SOCs) on the plasma membrane to open

the depletion of Ca stores inside the ER sends signal up to plasma membrane

• ER membrane MOVES UP to interact directly with channel on plasma membrane to open it

• Extracellular Ca rushes in to refill the ER

What physiological condition can occur in T-cells if SOC activation fails?

immunodeficiency

what regulated responses do Gq, Gs, and Gi GPCR carry out

• Gq INCreases Ca2+

• Gs INCreases cAMP

• Gi DECreases cAMP

• diff G proteins plug in to diff receptors to give distinct signal functions

steps of Gi pathway (4)

1. inhib. NT (dopamine) binds to GPCR

2. Gi-alpha is activated by GEF, separates to bind and inhibit adenylyl cyclase

3. decrease cAMP, decrease PKA activity

4. Gi-beta gamma subunits can also bind to ion channels like K+ channels to polarize cell (inhib membrane responsivity)

what is the primary cellular effect of activating presynaptic Gi-coupled receptors

it decreases cAMP and opens K+ channels, causing K+ efflux, hyperpol, and inhibited neurotransmitter release

how does activating postsynaptic Gs-coupled receptors lead to enhanced excitability

increases cAMP, activating PKA, which phosphorylates cation channels to allow Na+ influx (depol)

what NTs bind to adrenergic receptors

epinephrine and norepinephrine

what receptors are adrenergic and what G pathway are they involved in

• α1 (Gq blood vessel contraction)

• α2 (Gi brain relaxation)

• β1,2,3 (Gs increase cAMP)

what receptors are muscarinic and what do they do

• M1,3,5 (Gq excitatory, activate PKC)

• M2,4 (Gi lower cAMP)

what do M1, M3, M5 muscarinic receptors do

• couple to Gq to release Ca2+ from ER

• activate PKC with DAG and Ca2+

what do M2, M4 muscarinic receptors do

• couple to Gi to decrease cAMP

• decrease PKA activity and inhibit Ca2+ channels

what do α1 adrenergic receptor do

• couple to Gq to activate PLC that converts to IP3 and DAG

• IP3 releases Ca2+ that activate PKC with DAG

what do α2 adrenergic receptors do

• couple to Gi that inhibits adenylyl cyclase, DECrease cAMP

• decrease PKA activity and inhibit Ca2+ channels

• INCrease K+ conductance - decrease excitation

what do β1-3 adrenergic receptors do

• couple to Gs to increase cAMP

• increase PKA activity that increase Na+ and Ca2+ channels

explain the actions of α1 adrenergic receptor

1. norepinephrine binds to α1 receptor

2. bound receptor activates Gq-alpha

3. activated Gq-alpha activates PLC

4. PLC cuts PIP2 to IP3 & DAG

5. IP3 induces ER Ca2+ release

6. DAG + Ca2+ binds to activate PKC

7. Ca2+ release and activated PKC cause smooth muscle CONSTRICTION in blood vessels (sympathetic vasoconstriction)

how do adrenergic and muscarinic GPCR oppose each other in cardiac contraction

1. norepinephrine will bind to β1 receptor to activate Gs protein → activate PKA and Na+ and Ca2+ channels = sinoatrial contractions

2. acetylcholine will bind to M2 receptor to activate Gi protein → inhibit AC → decrease cAMP → inactivate Na+ Ca2+ channels = INHIBIT sinoatrial contractions

what is receptor desensitization

• reduce cell response to a receptor signal

• control signal duration to prevent abnormal cell response from excess signaling

what are two main ways a cell shuts off GPCR signaling

• blocking interaction between GPCR and its G-protein

• removing (internalizing) the receptor from the plasma membrane

how does G-protein-linked receptor kinase (GRK) and arrestin desensitize GPCR

1. GRK detects activated GPCR
2. GRK phosphorylates multiple sites on GPCR
3. arrestin is attracted to phosphorylated GPCR and binds
4. arrestin provides steric block -> G protein physically blocked from interacting with GPCR

how does arrestin shut down receptor signaling further

• arrestin internalizes GPCR (pulls GPCR into cell)
  - receptor is pulled in using endocytic vesicle
  - arrestin eventually falls off and GPCR is either degraded or recycled for future use by cell

how is the β adrenergic receptor desensitized

1. epinephrine binds to β-adrenergic receptor -> Gs-betagamma dissociates
2. Gs-betagamma recruits β-adrenergic receptor kinase (βARK) to phosphorylate receptor
3. β-arrestin (βarr) binds to phosphorylated receptor (BLOCKS signal)
4. receptor-arrestin is pulled inside cell by endocytosis
5. arrestin eventually dissociates and receptor is dephosphorylated or recycled back to cell surface

how does arrestin participate in g protein-independent signaling and what concept does this lead to in GPCR actions

• βarr serves as a "scaffolding" protein aka docking site for other proteins to bind to activate alternate signaling pathway

• happens after it pulls in GPCR by endocytosis

• leads to idea of balanced or biased agonism

what is G protein independent signaling

GPCR sends signals without using heterotrimeric G protein in its pathway

what happens to the traditional second messenger pathways (cAMP, Ca2+) when arrestin binds to the GPCR

they are BLOCKED bc arrestin physically blocks G proteins from interacting with the receptor

what is the difference between a balanced agonist and a biased agonist

• a balanced agonist EQUALLY activates both G-protein and β-arrestin pathways

• a biased agonist FAVORS activating one pathway over the other and ignores the other (G or βarr)

how is biased agonism applied to drugs like opiod receptors or GLP-1 receptors?

1. opiod receptors: find agonists that bind to opioid receptors with bias to amplify pain relieving effects (G-protein signaling) while avoiding addictive βarr effects

2. GLP-1R agonists biased towards G protein signaling to increase insulin secretion for type 2 diabetes

what is the μ-opioid receptor (MOR)

• GPCR that binds to opioids and is coupled to Gi protein

what are the 3 different signaling processes for the MOR (μ-opioid receptor)

1. G-protein-dependent (primary pain relief): Gi is activated to inactivate AC + activate K+ channel to reduce signaling for pain receptors

2. G-protein-independent (arrestin scaffold): receptor is phosphorylated and b-arrestin binds to act as scaffold and induce other signaling pathways

3. Desensitization (shutting down/tolerance): GRK phosphorylates receptor to recruit b-arrestin to decrease G protein signaling (cannot decrease pain sensitivity anymore)

what effect does the G protein pathway cause in MOR (μ-opioid receptor)

analgesia

what effect does the arrestin pathway cause in MOR (μ-opioid receptor)

respiratory depression, tolerance & dependence

what do tyrosine kinase linked receptors mediate and what ligands activates receptor tyrosine kinases

• growth control/responses

• peptide growth factors and hormones eg insulin, nerve growth factor

why are RTKs and Ras heavily represented in the oncogene family

their main function is to stimulate cell growth and proliferation, so overactivation of this pathway leads to cancer

what is the step by step pathway for a receptor tyrosine kinase (RTK) from ligand binding to cell growth

1. ligand binding & dimerization: peptide growt factor/hormone BIND to TWO RTK monomer on cell exterior → dimerization (come tgt)

2. autophosphorylation: kinase enzyme is built directly into RTK (unlike GPCRs)

   1. two receptors phosphorylate eo on their intracellular tail tyrosine residues

3. adapter & GEF docking: phosphorylated tyrosine act as docking sites for adapter protein (Grb2)

   1. adapter BRINGS a GEF (guanine nucleotide exchange factor; SOS) to membrane surface next to Ras

4. Ras activation: GEF swaps GDP for GTP on Ras (TURNS Ras ON)

5. MAP kinase cascade: active Ras trigger downstream kinase cascade (serine/threonine kinases) → activation

6. Cell growth response: active MAP kinase enter nucleus to change gene exp stimulating cell growth

what are GEFs and GAPs and how do they regulate the activity cycle of the small G-protein Ras

• GEF: guanine nucleotide exchange factors

  • promote exchange of GDP for GTP, turns inactive Ras-GDP to active Ras-GTP → on switch

• GAP: GTPase activating proteins

  • promote hydrolysis of bound GTP to GDP + Pi→ off switch

  • function: determine how long Ras stay active

what are small g proteins

single subunit monomeric proteins (20kd) such as Ras that are switched “on” for cell division by binding to GTP

why do mutations that block GAP activity or impair Ras’s GTPase function cause cancer

Ras cannot hydrolyze GTP to turn itself off, leading to permanently active Ras and continuous cell growth signaling

what are the step by step events needed following Ras activation - MAPK/Erk kinase cascade (3)

1. Ras-GTP activates the first kinase, MAPKKK (Raf) through protein-protein interaction

   1. Serine/Threonine kinase

   2. done by direct binding

2. Raf phosphorylates and activates MAPKK (MEK) -dual-specificity Ser/Thr + Tyr kinase

3. MEK phosphorylates on Thr and Tyr residues to make MAPK (Erk)

why are proteins in the RTK → Ras→MAPK pathway considered potential oncogenes

bc this pathway directly controls cell proliferation; overactivating mutations cause uncontrolled cell growth

what is the general mechanism of nuclear receptor and how does this work

they diffuse across the membrane

• ligands/agonists have a hydrophobic (lipid-soluble) character

• bc they are hydrophobic, they diffuse directly across plasma membrane w/o needing a surface receptor

where do nuclear receptors usually reside in and what general function do they perform

inside the cell (cytoplasm or nucleus), where hormones binds to its specific intracellular receptor protein

• once bound by a ligand, it acts as transcription factors to regulate gene expression (mRNA transcription)