GPCR Signaling - Lecture 7 Notes

GPCR Signaling – Lecture 7 Notes

  • Overview of GPCRs

    • G-Protein Coupled Receptors (GPCRs) are a large family of cell-surface receptors that transduce signals via heterotrimeric G proteins.
    • 7 transmembrane helices (7TM); serpentine receptors that pass the membrane seven times.
    • There are >20,00020{,}000 GPCRs (7TM receptors) with diverse ligands: hormones, neurotransmitters, photons, etc.
    • Approximately one-third of therapeutic drugs target GPCRs or related signaling pathways.
    • Structure-function insight: Rhodopsin and the β2-adrenergic receptor (β2-AR) share ligand-binding mechanism features; structural studies revealed GPCR–G protein coupling interfaces.

  • GPCR Structure and Activation

    • GPCRs have an exoplasmic (outside) ligand-binding face and a cytosolic face that couples to G proteins.
    • Ligand binding induces conformational changes that enable coupling to heterotrimeric G proteins.
    • Nobel Prize (2012): Lefkowitz and Kobilka recognized for determining active and inactive structures of β2-adrenergic receptor.
    • Activation cycle basics:
    • Inactive G protein: Ga–GDP bound; By dimer bound to GDP.
    • Hormone binding to GPCR promotes exchange of GDP for GTP on Ga (Ga–GTP) via a Guanine Nucleotide Exchange Factor (GEF) effect from the receptor.
    • Ga–GTP dissociates from By, allowing Ga to regulate effector proteins (e.g., adenylyl cyclase, PLC-β).
    • Ga’s intrinsic GTPase activity hydrolyzes GTP to GDP, Ga–GDP reassociates with By, returning to the inactive heterotrimer.
    • G proteins are heterotrimers with subunits: a-subunit (Ga, ~45$-$47 kDa), By dimer (β ~3535 kDa, γ ~7$-$9 kDa).
    • GEFs and GDIs regulate GDP–GTP cycling:
    • GEFs promote GDP release and GTP binding (activation).
    • GDIs help maintain GDP bound state in some contexts.
    • GPCRs can couple to different Ga subtypes to trigger distinct downstream responses.

  • G Proteins and Signal Transduction

    • Ga subunits define signaling outputs; common families include:
    • Gas (Gs): activates adenylyl cyclase → increases cAMP.
    • Gai (Gi): inhibits adenylyl cyclase → decreases cAMP.
    • Gaq (Gq): activates phospholipase C-β (PLCβ) → IP3 and DAG generation.
    • Golf, Go (other Ga family members) with various effectors.
    • Gβγ dimer (By) also participates in signaling, regulating ion channels and other targets.
    • Core consequence: two major second messenger pathways arise from GPCR activation:
    • cAMP pathway via adenylyl cyclase (Gs family).
    • IP3/DAG pathway via PLCβ (Gq family) and subsequent Ca²⁺ signaling and PKC activation.
    • Universal role: G proteins regulate many cellular processes beyond adenylyl cyclase, including phospholipases, ion channels, and cytoskeletal regulators; they are central to sensory systems (vision, olfaction).
    • Diversity: over 2121 distinct Ga subunits, multiple By subunits, and several Gy subunits exist in humans.

  • Signaling via cAMP and PKA

    • Gas activates adenylyl cyclase → converts ATP to CAMP:
    • ext{ATP}
      ightarrow ext{cAMP} + ext{PP}_i
    • cAMP activates Protein Kinase A (PKA).
    • PKA phosphorylates target proteins and transcription factors such as CREB (cAMP response element-binding protein).
    • CREB phosphorylation leads to gene transcription changes via CRE (cAMP response element).
    • Integrated epinephrine example: β-adrenergic receptor (a 7TM GPCR) coupled to Gas increases cAMP, activating PKA and altering both metabolic and gene expression programs.

  • Signal Amplification in GPCR Pathways

    • A single receptor–hormone complex can activate many Ga proteins, each Ga–GTP activating multiple adenylyl cyclases.
    • Thus, one ligand-receptor interaction yields a large amplification of the second messenger (e.g., hundreds to thousands of cAMP molecules per activated receptor).

  • GPCR Signaling via Phospholipase C-β (Gq pathway)

    • Gq family activates PLC-β at the plasma membrane.
    • PLC-β cleaves PI(4,5)P₂ (phosphatidylinositol 4,5-bisphosphate) to generate two second messengers:
    • Inositol 1,4,5-trisphosphate (IP₃)
    • Diacylglycerol (DAG)
    • IP₃ diffuses through cytosol to the endoplasmic reticulum (ER) and binds IP₃ receptors, opening Ca²⁺ channels and releasing Ca²⁺ into cytosol.
    • DAG remains in the membrane and, with Ca²⁺ and phosphatidylserine (PS), activates Protein Kinase C (PKC).
    • PKC translocates to the membrane upon activation and phosphorylates various substrates.
    • DAG can be cleaved to release arachidonic acid, contributing to eicosanoid signaling and inflammatory responses (target of NSAIDs among others).

  • G Proteins and Receptor Desensitization / Regulation

    • RGS proteins (Regulators of G protein signaling) accelerate GTP hydrolysis on Ga, shortening the active state and contributing to desensitization.
    • By dissociation and Gβγ signaling contribute to rapid modulation of signaling;
    • Desensitization may also involve receptor phosphorylation by GRKs and arrestins (not detailed in the slides but commonly discussed in GPCR signaling).

  • Structure and Activation Details for the β₂-adrenergic Receptor (β₂-AR)

    • The receptor contains seven transmembrane helices; ligand binding induces conformational changes especially in TM5 and TM6 and the C3 loop, creating a surface for Ga binding.
    • Activation causes the Gα subunit to undergo conformational changes, lengthening the α5 helix and forming an extended surface to contact the receptor cytoplasmic face.
    • GDP release from Ga occurs upon receptor interaction; GTP binding promotes dissociation of Ga from the By dimer and the activation of downstream effectors.
    • The Gα–GTP interaction with effectors like adenylyl cyclase leads to cAMP production; Gβγ can regulate other targets such as ion channels.

  • G Protein Subunit Switches and Monomeric GTPases

    • Monomeric GTPases (e.g., Ras) switch between active (GTP-bound) and inactive (GDP-bound) states via switches I and II in the protein structure.
    • Activation: A guanine nucleotide exchange factor (GEF) promotes GDP release; intracellular GTP binds, activating the protein.
    • Inactivation: GTPase activity hydrolyzes GTP to GDP and Pi, returning to the inactive state.
    • Switch I and Switch II are stabilized by interactions with the γ-phosphate of GTP; GDP-bound state relaxes to inactive conformation.
    • In Ras and heterotrimeric G proteins, these switches govern binding to downstream effectors.
    • GTPase-activating proteins (GAPs) and Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis, shortening signaling durations; regulation is often responsive to extracellular cues.

  • FRET (Fluorescence Resonance Energy Transfer) in GPCR Signaling

    • FRET is a method to study protein–protein interactions in live cells; requires two fluorophores in proximity (~1−10extnm1-10 ext{ nm}).
    • Donor: CFP (emisison ~extλ<em>1extaround490extnmext{λ}<em>1 ext{ around } 490 ext{ nm}) transfers energy to acceptor YFP (emission ~extλ</em>2extaround527extnmext{λ}</em>2 ext{ around } 527 ext{ nm}) if in close proximity.
    • Energy transfer efficiency depends on distance; increased FRET indicates interaction, decreased FRET indicates dissociation.
    • In GPCR signaling, FRET detects Gα–Gβγ dissociation upon receptor activation.
    • Real-time use: FRET reveals rapid (> seconds) G protein cycling after ligand binding.
    • Common visualization: CFP/YFP pair and corresponding emission/excitation wavelengths; fusion proteins track Gα–Gβγ proximity.

  • Practical GPCR Signaling Concepts and Examples

    • Epinephrine signaling via β-adrenergic receptors (β-ARs):
    • Epinephrine binds β-AR (a GPCR) → activation of Gas → adenylyl cyclase → ↑cAMP → PKA activation → CREB phosphorylation → nucleus gene expression changes.
    • Physiological outcomes include increased heart rate, glycogenolysis, lipolysis, and vascular/airway dynamics for ‘fight-or-flight’ responses.
    • 7TM receptor family and pharmacology:
    • >20,00020{,}000 receptor types; targets of about one-third of drugs.
    • Structural studies of rhodopsin and β₂-AR illuminate ligand-binding and receptor–G protein coupling interfaces.
    • Orphan GPCRs:
    • Orphan receptors are GPCRs whose endogenous ligands are unknown; numbering around ext 150ext{~}150; represent significant targets for drug discovery due to unknown signaling pathways and potential therapeutic interventions.
    • GPCR families and classic signaling outputs (selected examples):
    • Gs family: activates adenylyl cyclase → ↑cAMP.
    • Gi/Go family: inhibits adenylyl cyclase; modulates other targets such as ion channels.
    • Gq family: activates PLCβ → IP3/DAG → Ca²⁺ signaling and PKC activation.
    • Transducin (Gt) in vision: affects cGMP phosphodiesterase and phototransduction.

  • Key Tables and Receptor–Effector Relationships (conceptual summaries)

    • Major G protein classes and their effectors (illustrative examples):
    • Gs: activates adenylyl cyclase → ↑cAMP.
    • Gi: inhibits adenylyl cyclase → ↓cAMP.
    • Golf: promotes adenylyl cyclase in olfactory neurons; coupling to specific receptors.
    • Gq: activates PLCβ → IP3 and DAG.
    • Go: activates K⁺ channels; modulates Ca²⁺ channels.
    • Four major families of heterotrimeric G proteins (illustrative overview):
    • Gs family (Gas, Golf): stimulate adenylyl cyclase; ↑cAMP.
    • Gi/Go family (Gai, Go): inhibit adenylyl cyclase; modulate ion channels.
    • Gq family (Gaq): activate PLCβ; ↑IP3 and DAG.
    • G12/13 family (G12/13): influence Rho family GTPases; regulate cytoskeleton and cell movement.
    • GPCR examples by receptor type and downstream effectors:
    • β-adrenergic receptor (epinephrine): Gas → adenylyl cyclase → cAMP → PKA.
    • α₂-adrenergic receptor: Gai → ↓cAMP.
    • Muscarinic acetylcholine receptor (M2): Gi → ↓cAMP, often affects K⁺ channels via Gβγ.
    • Rhodopsin (vision) and odorant receptors: Gt family → PDE and cGMP signaling in sensory cells.

  • Signaling in Real Cells: Integration and Effects

    • Epinephrine orchestrates systemic responses: energy mobilization (glycogen breakdown, fat breakdown), heart rate increase, and redirecting blood flow during stress.
    • Crosstalk: Gβγ subunits can influence ion channels (e.g., K⁺ channels) and other signaling cascades in parallel with Ga pathways.
    • Second messengers and downstream targets can converge on protein kinases (PKA, PKC) and transcription factors (CREB), altering metabolism and gene expression.

  • Practice Questions (selected from the transcript)

    • Q1. Which of the following accurately describes a receptor that operates through non-receptor tyrosine kinases and is involved in the activation of immune cells? Options: A. MHC-peptide complex → T-cell receptor; B. Acetylcholine receptor (muscarinic) → GPCR; C. Fibroblast growth factor receptor → RTK; D. Insulin receptor → Ser/Thr kinase.
    • Q1 (signaling-pathway follow-up): Which signaling pathway is correct for cytokine receptors regulating hematopoietic stem cell differentiation? Options: A. PI3K → Akt → survival; B. JAK → STAT → gene expression; C. Ras-MAPK → gene transcription; D. PLC → DAG/IP3 → Ca²⁺ release.
    • Q2. Which combination of receptors is linked by modulation of neuronal synaptic transmission and uses biogenic amines as ligands? Options: A. NMDA and Adenosine receptor; B. Dopamine receptor and Serotonin receptor; C. Nicotinic ACh receptor and IL-8 receptor; D. Endothelin receptor and Oxytocin receptor.
    • Q3. Receptor–ligand pair primarily involved in vasoconstriction and smooth muscle contraction via GPCR mechanisms? Options: A. VIP receptor – intestinal secretion; B. Oxytocin receptor – uterine contraction; C. Endothelin receptor – vasoconstriction; D. Glucagon receptor – glycogenolysis.
    • Q4. Receptors that couple to serine/threonine kinase pathways? Options: A. BMP receptor; B. PDGF receptor; C. IL-6 receptor; D. EGFR.
    • Q5. Which receptors participate in Ras-MAP kinase signaling pathway? Options: A. Insulin receptor; B. PDGF receptor; C. FGFR; D. ACh (muscarinic) receptor; E. TGF-β receptor; F. EGFR. (From the slides—select combinations: e.g., 3, 4, 5, 6, etc.)
    • Q6. Which receptors mediate cytoplasmic steroid receptor signaling? Options: A. Glucocorticoid receptor, Estrogen receptor, Thyroid hormone receptor, Parathyroid hormone receptor, Progesterone receptor, Melanocyte-stimulating hormone receptor; B. 2,3,4,6; C. 1,2,5,6; D. 3,4,5,6.
    • Q7. Which of the following receptors function through the serine/threonine kinase pathway? Options: A. BMP receptor; B. Activin receptor; C. TGF-β receptor; D. IL-1 receptor; E. FGFR; F. EPO receptor. (From the slide: BMP, Activin, TGF-β, FGFR, EPO receptor listed in ser/thr kinase signaling.)
    • Q8. New scenario: Gα–CFP and Gβ–YFP fusion proteins in Dictyostelium plotted with cAMP addition; which statements are correct? Options: A. 1, 2, 3; B. 1, 3, 4; C. 2, 3, 4; D. 1, 4 only.
    • Q9. In the recombinant epinephrine receptor–acetylcholine receptor construct, which statements are correct about cAMP and receptor coupling? Options: A. 1 and 3 only; B. 1, 3, 4 only; C. 2 and 4 only; D. 1, 2, 3 only.
    • Q10. Part-C: Dictyostelium cAMP signaling fusion-protein study results interpretation—multiple statements about Gα–Gβ–γ associations and dissociation after cAMP exposure.
    • These questions illustrate key concepts: GPCR activation, G protein cycling, second messengers, receptor coupling, and experimental tools like FRET.

  • Connections to Foundational Principles and Real-World Relevance

    • GPCR signaling exemplifies how extracellular ligands translate into rapid intracellular responses via second messengers and kinases.
    • The diversity of Ga subunits and effectors enables tissue- and context-specific responses to the same ligand (e.g., epinephrine affects liver, adipose, heart via different Ga couplings).
    • Understanding GPCR structure–function relationships informs drug design and pharmacology; many therapeutic drugs target GPCRs or downstream pathways.
    • Techniques such as FRET provide dynamic, real-time readouts of molecular interactions, allowing direct observation of G protein cycling and receptor activation.

  • Formulas and Key Reactions (LaTeX)

    • Adenylyl cyclase reaction: ext{ATP}
      ightarrow ext{cAMP} + ext{PP}_i
    • GTP hydrolysis on Ga: ext{Ga-GTP}
      ightarrow ext{Ga-GDP} + ext{P}_i
    • PLCβ reaction on PI(4,5)P₂: ext{PI(4,5)P}2 ightarrow ext{IP}3 + ext{DAG}
    • IP₃-mediated Ca²⁺ release from ER and DAG-mediated PKC activation: Ca²⁺ flux and PKC translocation depend on these messengers.
    • cAMP-dependent transcription: cAMP → PKA activation → CREB phosphorylation → CRE-mediated gene expression.

  • Summary of Core Concepts

    • GPCRs transduce signals via Ga–By heterotrimers; GDP/GTP cycling regulates activation/inactivation.
    • Two principal second-messenger pathways: cAMP/PKA (Gas) and IP₃/DAG/Ca²⁺ (Gq).
    • Gβγ signaling modulates ion channels and other effectors; Gα subunits confer specificity to downstream pathways.
    • Receptor activation is followed by amplification, leading to robust cellular responses.
    • Structural biology and real-time imaging (e.g., FRET) deepen understanding of receptor–G protein interactions and signaling dynamics.

  • References to Key Concepts and Figures (from the transcript content)

    • 7TM receptors and the structural basis for ligand binding and G protein coupling (β₂-AR and rhodopsin as models).
    • Activation mechanism: GDP release, GTP binding, α subunit dissociation, and effector engagement.
    • Ras/MAPK signaling context as part of broader GTPase regulation (monomeric GTPases).
    • Orphan GPCRs as potential drug targets due to unknown endogenous ligands and signaling pathways.
    • FRET-based visualization of G protein dissociation and GPCR cycling in live cells.

  • Notes on Terminology and Abbreviations

    • GPCR: G-Protein Coupled Receptor
    • Ga: G protein alpha subunit
    • By: G protein beta/gamma dimer
    • GEF: Guanine-Nucleotide Exchange Factor
    • GDP/GTP: Guanine diphosphate/triphosphate bound states
    • PLCβ: Phospholipase C-β
    • IP₃: Inositol 1,4,5-trisphosphate
    • DAG: Diacylglycerol
    • PKA: Protein Kinase A
    • CREB: cAMP Response Element-Binding protein
    • CRE: cAMP Response Element
    • PKC: Protein Kinase C
    • eicosanoids: signaling lipids including prostaglandins

  • Formatting notes

    • All numerical references and key reactions are presented in LaTeX as …… where appropriate.
    • The notes are structured as bullet points under top-level headings to reflect the slide-by-slide content in the transcript.