Signal Transduction II: Neurotransmitter-Gated Ion Channel Receptors and GPCR Signaling

Neurotransmitter-Gated Ion Channel Receptors

• Activation is determined by whether the neurotransmitter is excitatory or inhibitory.
  • Excitatory neurotransmitters promote opening of cation-permeable channels, leading to depolarization.
  • Inhibitory neurotransmitters promote opening of anions (e.g., Cl⁻) channels, promoting hyperpolarization.
• Oligomeric structure and gating concept
  • Receptors are multimeric; subunit composition defines channel properties.
  • Gating: ligand binding at the extracellular domain induces conformational changes that open the ion channel pore to allow ion flux.
• Signal termination strategies
  • Inactivation vs deactivation distinguishes termination mechanisms (details depend on channel type and receptor subtype).
• Role at synapses
  • Critical in signaling at nerve–nerve and nerve–muscle synapses.
  • Presynaptic and postsynaptic elements coordinate neurotransmitter release and receptor activation.
• Example: P2X receptors
  • Extracellular ATP acts as an excitatory neurotransmitter for the P2X family of ligand-gated ion channels.
• Core concept: ligand-gated ion channels as receptors
  • Bind neurotransmitters extracellularly and form an ion channel that directly changes the postsynaptic membrane potential.

Structure and gating principles of ligand-gated ion channel receptors

• Receptors are oligomeric complexes with diverse subunit stoichiometries:
  • Pentamer (e.g., nicotinic ACh receptors in many contexts)
  • Tetramer
  • Trimer
• Major receptor families and representative examples
  • Cys-loop receptor superfamily: nicotinic ACh, 5-HT₃, GABA_A, glycine, etc.
  • Glutamate receptor family: NMDA, AMPA, kainate (not all are Cys-loop receptors)
  • P2X receptor family (ATP-activated)
  • ZAC receptors (zinc-activated channels)
• Common architectural motifs
  • Receptors are generally extracellular N-termini with ligand-binding domains and transmembrane channel domains.
  • Pentameric or tetrameric assemblies form the ion-conducting pore.
• Example wiring: nicotinic ACh receptor
  • A representative model is the nicotinic ACh receptor of adrenal gland-linked metabolic regulation.

Receptors at the synapse: rest and activation dynamics

• Resting state: presynaptic terminal contains neurotransmitter-filled vesicles; voltage-gated Ca²⁺ channels are closed; synaptic vesicles dock at the active zone.
• Action potential arrives: voltage-gated Ca²⁺ channels open; Ca²⁺ influx triggers transmitter release into the synaptic cleft.
• Neurotransmitter binding to postsynaptic ligand-gated ion channels opens the channel, allowing ion flux and generating an electrical signal in the postsynaptic cell.
• Termination of signaling involves clearance/removal of neurotransmitter and desensitization of receptors where appropriate.

Excitatory vs inhibitory synaptic signaling (summary)

• Excitatory synapse
  • Activation leads to Na⁺ influx, depolarizing the membrane and increasing the likelihood of action potential firing.
• Inhibitory synapse
  • Activation leads to Cl⁻ influx, hyperpolarizing or stabilizing the membrane and decreasing the likelihood of action potential firing.

General ligand-gated ion channel families: structural overview

• Major families in the ligand-gated ion channel realm include:
  • Cys-loop receptor superfamily (nicotinic ACh, 5-HT₃, GABA_A, glycine, etc.)
  • Glutamate receptor family (NMDA, AMPA, kainate)
  • P2X receptor family (ATP-activated)
  • ZAC receptors
• Common structural organization: multiple subunits assemble into an oligomeric channel with a central pore.

The nicotinic acetylcholine receptor (nAChR) as a paradigm

• Structure and function
  • Example: nicotinic ACh receptor of adrenal gland; ligand binding results in channel opening and cation flux.
• Relevance to signaling: fast synaptic transmission via direct ion flux across the postsynaptic membrane.

G Protein-Coupled Receptors (GPCRs)

• GPCRs encode a substantial portion of the human genome and biology
  • ~3% of the human genome encodes GPCRs (≈ 3 ext{%} of ≈ 25{,}000 genes).
  • ~850 unique GPCRs exist in humans.
  • Approximately 50 ext{%} of current therapeutic drugs target GPCRs (e.g., beta-blockers in cardiovascular medicine).
• Nature and diversity
  • GPCRs are a biologically diverse group whose ligands range from large glycoproteins and small peptides to amino acids, nucleotides, lipids, and other molecules.
• Historical significance
  • Lefkowitz and Kobilka contributed foundational insights leading to Nobel Prizes: G-proteins and GPCR signaling (Lefkowitz, 2012; Kobilka, 2012).
  • 🧭 Structural milestones include the first crystal structure of a GPCR (rhodopsin) by Palczewski et al. (2000) and ongoing comparative structural analyses.

Heterotrimeric G proteins: composition and function

• Composition
  • Trimeric complex: α, β, and γ subunits.
  • α subunit: largest (≈ 40-50\,kDa); site of guanine nucleotide binding; has intrinsic GTPase activity; ≈ 20 unique α-subunits.
  • β subunit (≈ 35\,kDa) and γ subunit (≈ 15-20\,kDa) form a tightly associated membrane-associated dimer.
• Activation cycle
  • In the resting state, the heterotrimer is bound to GDP on the α-subunit and is associated with the receptor.
  • Upon receptor activation, GDP on α is exchanged for GTP, causing dissociation of Ga-GTP from the Gβγ dimer.
  • Both Ga-GTP and Gβγ can interact with downstream effectors.
• Key concept: amplification
  • GPCR signaling involves multiple amplification steps; a single activated receptor can trigger many downstream events.
  • Example: visual signaling where a single photon-activated rhodopsin can activate about ext{700-500} transducin G proteins (≈ 500 per photon) depending on context.

Major steps in GPCR signaling cycles

• Activation steps (receptor-G protein coupling)
  • Ligand binds GPCR → receptor undergoes conformational change → GDP on Ga replaced by GTP → Ga dissociates from Gβγ.
  • Ga-GTP and Gβγ activate downstream effectors such as adenylyl cyclase or phospholipase C (PLC).
• Inactivation and reassembly
  • Ga has intrinsic GTPase activity hydrolyzing GTP to GDP, leading to reassembly of the inactive heterotrimer with Gβγ.
• Structural insights
  • Crystal structures reveal how GPCRs interact with G proteins, with conformational changes in helices and loops (TM5, TM6, etc.) that govern activation.

Two major second messenger cascades downstream of GPCRs

• Cyclic AMP (cAMP) pathway (Gs and Gi families)
  • Activation of adenylyl cyclase (AC) by Gs increases the conversion of ATP to cyclic AMP (cAMP).
  • cAMP activates protein kinase A (PKA), leading to phosphorylation of target proteins and downstream effects.
  • Gi-type G proteins inhibit AC, reducing cAMP production.
• Inositol trisphosphate (IP₃) / Ca²⁺ pathway (Gq family)
  • Activation of phospholipase C-β (PLC-β) by Gq leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) into IP₃ and diacylglycerol (DAG).
  • IP₃ triggers Ca²⁺ release from the endoplasmic reticulum via IP₃-gated Ca²⁺ channels; DAG activates protein kinase C (PKC) in the presence of Ca²⁺.
• Schematic overview (from slides)
  • Receptors → G protein activation (Ga-GTP, Gβγ) → downstream effectors (AC, PLC) → second messengers (cAMP, IP₃, DAG, Ca²⁺) → kinases like PKA and PKC → gene expression and metabolic outcomes.

GPCR signaling amplification and transcriptional regulation

• Amplification in GPCR signaling (an extreme example)
  • Visual signaling: a single photon can activate ~500 transducin G proteins, illustrating powerful amplification.
• cAMP-driven transcriptional regulation
  • cAMP activates PKA, which phosphorylates transcription factors such as CREB.
  • CREB phosphorylation leads to CRE-mediated gene expression changes via CREB-binding protein (CBP).
• Downstream consequences include energetic and metabolic changes in numerous tissues, as captured in tissue-specific tables of responses to CAMP elevations.

Desensitization and termination of GPCR signaling

• Desensitization concept
  • The receptor remains on the cell surface but becomes functionally uncoupled from G protein activation.
• Desensitization mechanisms
  • Covalent phosphorylation of the receptor by receptor-specific kinases (GRKs) or second messenger-regulated kinases (PKA/PKC).
  • Arrestin binding to phosphorylated receptors (β-arrestin) and subsequent receptor internalization into endosomes.
  • Receptors can recycle back to the plasma membrane or be targeted for degradation.
• Signaling diversity and arrestin-biased signaling
  • Arrestin can scaffold alternative signaling pathways, enabling arrestin-dependent signaling independent of G proteins.
• The concept of biased agonism
  • Biased agonists stabilize receptor conformations that preferentially signal through either G proteins or arrestin pathways; the idea is to separate therapeutic effects from adverse effects by selecting pathway bias.
  • Illustrative schemes show biased ligands interacting with GRKs and arrestin to direct signaling outcomes toward either G protein or arrestin pathways.

GPCR structure and conformations: what structural biology reveals

• Aligned crystal structures of four diverse GPCRs
  • Rhodopsin, β₂-adrenergic receptor (β₂AR), adenosine A₂A receptor, and another GPCR (BAR) demonstrate conserved framework with variations in extracellular loops and intracellular sides.
  • Key observation: extracellular loops and transmembrane helices (e.g., TM5, TM6) rearrange to accommodate active vs inactive states; some receptors show outward movement of TM6 upon activation.
  • The terms Out vs In describe TM6 orientation in different receptor states, illustrating activation geometry.
• Structural insights into GPCR-G protein coupling
  • The cytoplasmic view highlights interaction surfaces with G proteins and the role of specific helices in coupling efficiency and specificity.
• Notable historical structures
  • Rhodopsin: first GPCR crystal structure (Science, 2000).
  • Subsequent GPCR structures (β₂AR, adenosine A2A, etc.) expanded understanding of activation mechanisms and biased signaling.
• Significant figures and researchers
  • Alfred G. Gilman and Martin Rodbell: Nobel Prize (1994) for discovery of G-proteins and their role in signal transduction.
  • John H. Hord and Krzysztof Palczewski: contributions to GPCR structure; Palczewski et al. reported the crystal structure of rhodopsin (Science, 2000).
  • Lefkowitz and Kobilka: Nobel Prize (2012) for discoveries related to GPCRs and their mechanisms.

Key signaling players and pathways: detailed snapshots

• G protein cycle (illustrative snapshots)
  • Inactive receptor with GDP-bound Ga associates with Gβγ.
  • Agonist binding reorients the receptor to promote GDP-GTP exchange on Ga.
  • Ga-GTP dissociates from Gβγ and activates downstream targets (e.g., AC or PLC).
  • Ga hydrolyzes GTP to GDP, reassociates with Gβγ, returning to the inactive state.
• Adenylyl cyclase and CAMP signaling
  • Activation by Gs increases [cAMP]; Gi inhibits adenylyl cyclase to reduce [cAMP].
  • cAMP activates PKA; PKA phosphorylates target proteins to generate cellular responses.
• Phosphodiesterases (PDEs)
  • PDEs degrade cAMP, terminating the signal.
• IP₃/DAG and PKC signaling
  • PLCβ-mediated cleavage of PI(4,5)P₂ yields IP₃ and DAG.
  • IP₃ mobilizes Ca²⁺ from ER stores; DAG activates PKC in coordination with Ca²⁺.
• Ca²⁺ homeostasis and signaling cross-talk
  • ER Ca²⁺ stores release Ca²⁺ via IP₃ receptors; Ca²⁺ regulates enzymes and transcription factors.
  • Ca²⁺ homeostasis involves ER Ca²⁺ pumps (SERCA), plasma membrane Ca²⁺ pumps, Na⁺/Ca²⁺ exchangers, and mitochondrial uptake.

The bigger picture: signal transduction via IP₃ and DAG pathways

• IP₃-DAG-PKC axis represents a major route by which GPCRs regulate non-genomic and genomic responses.
• The IP₃ receptor-mediated Ca²⁺ release interfaces with Ca²⁺-dependent kinases and phosphatases to shape multiple cellular processes.
• DAG and Ca²⁺ together coordinate PKC activation and downstream phosphorylation events that modulate metabolism, gene expression, and cell fate decisions.

Clinical relevance and applications

• GLP-1 receptor signaling and diabetes therapy
  • TrulicityⓇ exemplifies a GPCR-targeted therapy for Type 2 Diabetes by activating the glucagon-like peptide-1 receptor (GLP-1R).
  • GPCR-targeted therapies comprise a large portion of modern pharmacology.
• Hormonal and metabolic regulation via GPCRs and downstream effectors
  • Hormonal regulation of insulin, glucagon, and related metabolic pathways can involve GPCR signaling networks that influence substrate utilization, secretion, and tissue responses.

Additional notes and cross-links

• Terminology crosswalk
  • Receptors: ligand-gated ion channels vs GPCRs; both initiate signaling but via distinct mechanisms (direct ion flux vs second messenger cascades).
  • Desensitization vs internalization: desensitization uncouples receptor from G proteins; internalization pulls receptors into endosomes for recycling or degradation.
• Foundational figures and references (identify who contributed foundational ideas and structural insights):
  • Alfred G. Gilman & Martin Rodbell – G-protein signaling and signal transduction: Nobel Prize (1994).
  • Palczewski et al. – Crystal structure of rhodopsin (Science, 2000).
  • Lefkowitz & Kobilka – GPCR signaling and structure, Nobel Prize (2012).
• Practical experiments and topics to review
  • Understand how GPCRs couple to different G proteins (Gs, Gi, Gq) and the downstream effectors they engage (AC, PLC).
  • Recall the two major second messenger systems (cAMP/PKA and IP₃/DAG/PKC) and how they regulate cellular functions.
  • Be able to discuss biased agonism concepts and how ligands can preferentially trigger distinct pathways.

Quick reference figures and terms from the slides

• “Two major GPCR second messenger cascades: ext{cAMP} and ext{IP}_3/ ext{Ca}^{2+} signaling.”
• “Activation of adenylyl cyclase by Gs and inhibition by Gi”
• “PLCβ-generated IP₃ and DAG from PI(4,5)P₂”
• “Ca²⁺ homeostasis involving ER Ca²⁺ stores and IP₃ receptors”
• “Desensitization: GRKs, PKA/PKC phosphorylation, arrestin binding, receptor internalization”
• “Biased agonism: G protein-biased vs arrestin-biased signaling”
• “GPCR crystal structures: rhodopsin, β₂AR, adenosine A₂A, BAR; Out vs In conformations around TM helices”
• “Clinical example: GLP-1 receptor agonist Trulicity”