✅Lecture 1 - G-Protein-Coupled Receptors (GPCRs)
1. General Introduction to GPCRs
Definition: GPCRs are proteins embedded in the cell membrane that receive messages (signals) from external ligands.
Key Characteristics:
Largest family of cell-surface receptors in the human body.
Mediate a vast array of physiological responses.
The human genome encodes approximately 800 different GPCRs.
For many, the natural ligand is unknown; these are called orphan receptors.
Importance & Examples:
Muscarinic acetylcholine receptors: Regulate central and peripheral nervous system functions.
Histamine receptors: Mediate cellular response to histamine (e.g., allergic reactions).
Serotonin receptors: Involved in mood, memory, and other brain functions.
Glucagon-like peptide-1 receptors: Control blood sugar levels.
GABA-B receptors: Inhibitory, reduce action potentials.
Pharmacological Targets:
GPCRs are the targets for ~30% of all prescribed small-molecule drugs.
Also targets for many recreational drugs.
2. Diversity of Endogenous GPCR Agonists
Agonists are natural molecules that activate GPCRs. They can be categorized into classes:
Catecholamines (synthesized from tyrosine):
Examples: Adrenaline, Noradrenaline, Dopamine.
Biogenic Amines (synthesized from various amino acids):
Examples: Histamine (from histidine), Serotonin/5-HT (from tryptophan).
Peptides:
Examples: Endothelin, Bradykinin, Neuropeptide Y (NPY).
Glycoprotein Hormones:
Examples: Thyrotropin (TSH), Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH).
Lipids:
Examples: Prostaglandins, Leukotrienes, Anandamide (natural ligand for cannabinoid receptors).
Nucleotides/Nucleosides:
Examples: Adenosine, ADP, ATP, UDP, UTP.
Other:
Odorants: Activate odorant receptors in the nose.
Light: Activates the GPCR Rhodopsin in the retina.
3. Structure of GPCRs
General Structure:
A single polypeptide chain, typically 350-500 amino acids long.
Known as a seven-pass transmembrane receptor or serpentine receptor.
It has 7 transmembrane (TM) α-helical domains that span the membrane.
An extracellular N-terminus and an intracellular C-terminus.
3D Conformation:
The structure is not flat but a complex 3D shape. The helices create a binding pocket for ligands.
Ligand Binding:
Ligands (like noradrenaline) bind within the transmembrane helix bundle.
The receptor also has binding sites for the G-protein's α and γ subunits.
4. Classification of GPCRs
GPCRs are classified into three main families based on sequence and functional similarity:
Class A (Rhodopsin-like):
The largest class. Includes rhodopsin, adrenergic receptors, and many neurotransmitter receptors.
Class B (Secretin and Adhesion families):
Includes receptors for large peptide hormones like secretin, glucagon, and VIP.
Class C (Glutamate class):
Includes metabotropic glutamate receptors and GABA-B receptors. Often form dimers.
Binding Sites:
Orthosteric Site: The primary site where the endogenous agonist binds. Antagonists block this site.
Allosteric Site: A different site on the receptor. Binding by allosteric modulators can enhance or inhibit the effect of the orthosteric ligand.
5. GPCR Activation & The G-Protein Cycle
What happens after agonist binding?
The agonist binding induces a conformational change in the receptor.
This change enables the receptor to interact with and activate a heterotrimeric G-protein on the intracellular side.
Key regions for G-protein coupling are the 2nd and 3rd intracellular loops (i2, i3) and the C-terminal tail.
The Heterotrimeric G-Protein:
Composed of three subunits: Gα (binds GDP/GTP), Gβ, and Gγ.
The human genome encodes many variants: 21 Gα, 5 Gβ, 12 Gγ.
The Cycle (How the message is conveyed):
Resting State: G-protein is inactive, with GDP bound to the Gα subunit.
Activation: The agonist-bound GPCR acts as a Guanine Nucleotide Exchange Factor (GEF), causing Gα to release GDP and bind GTP.
Dissociation: The Gα-GTP complex dissociates from the Gβγ dimer. Both Gα-GTP and Gβγ can now activate downstream effector proteins.
Signal Termination: The intrinsic GTPase activity of the Gα subunit hydrolyses GTP to GDP.
Reassociation: Gα-GDP reassociates with Gβγ, reforming the inactive heterotrimer, ready for a new cycle.
6. G-Protein Families (α Subtypes) and Effector Pathways
Different Gα subunits define the major G-protein families and their distinct signaling pathways.
Gαs Family (Stimulatory):
Primary Effector: Adenylyl Cyclase (AC)
Mechanism: Gαs-GTP activates AC, which converts ATP to cyclic AMP (cAMP).
Downstream Cascade:
cAMP activates Protein Kinase A (PKA).
PKA phosphorylates metabolic enzymes and the transcription factor CREB, regulating energy metabolism and gene transcription.
Example GPCRs: β-adrenergic receptors, Glucagon receptor, Histamine H2 receptor.
Note: Gαolf is a variant in olfactory neurons for smell transduction.
Gαi/o/z Family (Inhibitory):
Primary Effector: Adenylyl Cyclase (AC) - but with an inhibitory effect.
Mechanism: Gαi-GTP inhibits AC, leading to a decrease in cAMP levels, antagonizing Gαs signaling.
Gβγ Signaling (Critical for this family): The released Gβγ dimer regulates:
Ion Channels: Activates K+ channels (GIRK) causing hyperpolarization, and inhibits Voltage-gated Ca2+ channels.
Other Effectors: Can activate PI3Kγ and recruit GRKs for receptor desensitization.
Example GPCRs: Muscarinic M2/M4, Adrenergic α2, Opioid receptors.
Gαq/11 Family:
Primary Effector: Phospholipase C-β (PLCβ)
Mechanism: Gαq-GTP activates PLCβ, which hydrolyzes the membrane lipid PIP2 into two second messengers:
IP3: Triggers Ca2+ release from the endoplasmic reticulum.
DAG: Remains in the membrane.
Downstream Cascade: The combined rise in Ca2+ and DAG activates Protein Kinase C (PKC). Ca2+ also activates Calmodulin (CaM).
Example GPCRs: α1-adrenergic, Histamine H1, Muscarinic M1/M3/M5 receptors.
Gα12/13 Family:
Primary Effector: RhoGEFs (Guanine nucleotide Exchange Factors).
Mechanism: Gα12/13-GTP activates RhoGEFs, which then activate the small GTPase RhoA.
Downstream Cascade: RhoA activates Rho-Kinase (ROCK), leading to cytoskeletal remodeling, smooth muscle contraction, and cell migration.
Example GPCRs: Receptors for thrombin, lysophosphatidic acid (LPA).
Gαt Family (Transducin - Specialized for Vision):
GPCR: Rhodopsin.
Primary Effector: Phosphodiesterase 6 (PDE6).
Mechanism: Light activates Rhodopsin, which activates Gαt. Gαt-GTP activates PDE6, which breaks down cGMP.
Downstream Cascade: The drop in cGMP causes cGMP-gated Na+/Ca2+ channels to close, hyperpolarizing the rod cell and generating a visual signal.
7. Gβγ Signalling
The Gβγ dimer is not just a passive spectator; it is a potent signalling molecule itself.
Effectors regulated by Gβγ include:
Ion Channels: Activates K+ channels, inhibits Ca2+ channels (primarily via Gi/o-coupled receptors).
Enzymes: Can activate or inhibit certain isoforms of Adenylyl Cyclase (AC2, AC4), PLCβ, and Phosphoinositide 3-kinase (PI3K).
Receptor Desensitization: Activates GPCR Kinases (GRKs like GRK2/3), which phosphorylate the receptor.
8. MAPK Pathway
Mitogen-Activated Protein Kinase (MAPK) pathways (e.g., ERK pathway) regulate cell proliferation, differentiation, and survival.
GPCRs can activate MAPK pathways through various mechanisms involving:
Gα subunits (e.g., Gαs, Gαi, Gαq)
Gβγ dimers
Arrestins (proteins involved in receptor desensitization).
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Questions/Tasks from Lecture
Name SIX classes of endogenous GPCR agonist and provide an example for each.
Answer provided in Section 2 above.
With the aid of a diagram explain the main structural difference between class A, B and C GPCRs.
While a diagram cannot be drawn here, the key differences are:
Class A: Typically have a small N-terminus; ligand binds within the transmembrane bundle.
Class B: Have a large, structured extracellular N-terminus that is critical for ligand binding (often for large peptides).
Class C: Have a very large N-terminus that forms a Venus flytrap module for ligand binding (e.g., glutamate); often function as dimers.
Explain the difference between orthosteric and allosteric binding sites.
Answer provided in Section 4 under "Binding Sites".
Describe the GPCR cycle.
Answer provided in Section 5 under "The Cycle".
Recommended read info:
1) Rang & Dale’s Pharmacology — Chapter 3 (pp.29–40) — Receptors and drug–receptor interactions
Overview
Introduces the concept of drug receptors as macromolecules (usually proteins) that mediate the effects of drugs.
Distinguishes intracellular vs cell-surface receptors; emphasizes most therapeutic drugs act at proteins in the plasma membrane.
Key concepts and definitions
Affinity (Kd): concentration of ligand at which half the receptors are occupied. Smaller Kd = higher affinity.
Efficacy: the ability of a bound drug to initiate a response (intrinsic activity). Agonists have efficacy > 0; antagonists have efficacy = 0.
Potency: dose/concentration required to produce a defined effect (often EC50). Potency depends on both affinity and efficacy.
EC50: concentration of agonist producing 50% of the maximal effect.
Spare receptors: presence of more receptors than needed to elicit a full response; explains why occupancy and response curves can differ.
Quantitative relationships
Law of mass action for reversible binding: R + A ⇌ RA. At equilibrium: Kd = [R][A]/[RA].
Fractional occupancy (f) = [A] / ([A] + Kd).
Operational model / receptor theory: links occupancy to response taking efficacy into account. Note that identical occupancy may give different responses for different agonists.
Dose–response curves
Graded (single system) vs. quantal (population) responses.
Log–dose plot: sigmoidal curve; shifting right = lower potency.
Competitive antagonists: shift agonist dose–response curve to the right (parallel) without reducing Emax (surmountable); can be overcome by increasing agonist.
Non-competitive antagonists / irreversible antagonists: reduce Emax (insurmountable) by reducing available receptor number or coupling efficiency.
Types of ligands
Full agonist: produces maximal response.
Partial agonist: produces submaximal response even at full receptor occupancy — can act as antagonist in presence of full agonist.
Inverse agonist: reduces constitutive activity of receptors that have basal activity.
Allosteric modulators
Bind to sites distinct from the orthosteric (agonist) site; can increase (positive) or decrease (negative) affinity/efficacy and can alter cooperativity.
Receptor heterogeneity and subtypes
Different receptor subtypes (e.g. adrenergic α, β) explain tissue-selective drug responses.
Clinical/experimental notes
Radioligand binding assays quantify affinity (Kd) and Bmax (receptor density).
Functional assays measure EC50 and Emax; combining binding and functional data distinguishes affinity vs efficacy changes.
2) Molecular Pharmacology: From DNA to Drug Discovery — Chapter 3 (pp.31–51)
(Focus: molecular cloning of drug targets and GPCRs / receptor molecular biology — concise notes highlighting molecular techniques and implications for drug discovery.)
Chapter focus
How DNA cloning and molecular biology revolutionized identification and characterization of drug targets.
Steps: isolation of mRNA, cDNA cloning, expression cloning, sequence analysis, heterologous expression and functional assays.
GPCR cloning and consequences
Cloning showed that many receptors share a 7-transmembrane (7TM) structural motif; revealed a superfamily of GPCR genes.
Sequence homology allowed classification into receptor families and prediction of ligand-binding residues.
Molecular techniques emphasized
Expression cloning: functional screens of cDNA libraries to find receptors for known ligands.
Site-directed mutagenesis: mapping ligand-binding sites and residues critical for activation (e.g., conserved DRY motif, TM6 movements).
Chimeric receptors: swapping domains between subtypes to identify regions conferring ligand specificity or G-protein coupling.
From sequence to pharmacology
Identification of receptor gene sequences enabled:
Development of recombinant expression systems for high-throughput screening.
Rational structure–activity relationship (SAR) studies using mutational data.
Discovery of orphan receptors and subsequent deorphanization (finding endogenous ligands).
Signalling diversity and regulation
GPCRs couple to multiple G-proteins (Gs, Gi/o, Gq/11) and can signal through β-arrestins (G-protein-independent pathways).
Concepts of agonist bias (ligand-directed signalling): different ligands stabilise different active states leading to pathway-selective signalling.
Drug discovery implications
Molecular cloning accelerated target-based screening and allowed use of cell-based functional assays for mechanistic readouts.
Identification of receptor subtypes enabled selectivity engineering to improve therapeutic index.
3) Molecular Biology of the Cell (Alberts) — Chapter 15 (pp.832–849) — Cell Signaling (focused pages)
(These pages are within the chapter on cell signalling; notes emphasise receptor mechanisms, second messengers, and intracellular cascades relevant to pharmacology.)
Principles highlighted
Signal reception and transduction: extracellular signals (ligands) are recognized by receptors; information is transmitted via conformational change and biochemical cascades.
Types of receptors: ion-channel-linked receptors, GPCRs (7TM), enzyme-linked receptors (RTKs), and intracellular receptors (steroid receptors).
GPCR mechanism recap
Agonist binding triggers conformational changes, promoting G-protein (heterotrimeric) activation: GDP→GTP on Gα, dissociation of Gα and Gβγ, and regulation of effectors (adenylyl cyclase, PLC, ion channels).
Second messengers: cAMP, IP3, DAG, Ca2+. Temporal and spatial control of these messengers is crucial for specificity.
Receptor tyrosine kinases (RTKs)
Ligand-induced dimerisation and trans-autophosphorylation create docking sites for SH2/PTB domain-containing adaptor proteins (e.g., Grb2), linking to Ras–MAPK, PI3K–Akt pathways.
Downstream cascades and amplification
Kinase cascades (MAPK) provide amplification and integration; phosphorylation often creates docking sites, altering localization/activity.
Desensitization and feedback
Negative feedback via phosphorylation (by GRKs for GPCRs or receptor-associated kinases for RTKs), receptor internalization, and phosphatases.
Scaffolding proteins organize signalling modules to enhance specificity and reduce crosstalk.
Cellular outcomes
Short-term effects: changes in enzyme activity, ion flux.
Long-term effects: gene expression changes via transcription factors (e.g., CREB downstream of cAMP/PKA), cell growth/survival decisions (via MAPK/PI3K).
4) Lefkowitz RJ (2004) — "Historical review: a brief history and personal retrospective of seven-transmembrane receptors" (Trends Pharmacol Sci. 25:413–422)
Core themes
Personal retrospective covering major milestones: adrenergic receptor purification, cloning, homology with rhodopsin, discovery of large GPCR gene family.
Key discoveries:
Evidence for receptor G-protein coupling and role of GTP in signal transduction.
Identification of GPCR kinases (GRKs) and β-arrestins as central to desensitization and receptor internalization.
Concept of receptor phosphorylation → arrestin binding → endocytosis and its role in resensitization vs down-regulation.
Mechanistic insights
Describes molecular basis for agonist-promoted desensitization and the interplay between phosphorylation, arrestin binding, endocytosis, and recycling vs degradation.
Notes on GPCR structure–function: how advances in cloning and structural biology revealed conserved motifs and activation movements (e.g., TM6 outward movement).
Broader impact
GPCRs are the largest family of drug targets; understanding their molecular biology enabled new therapeutic strategies (biased agonism, allosteric modulators).
Quick cross-chapter synthesis (what to remember)
Receptor theory (Rang & Dale) gives the pharmacological language (affinity, efficacy, potency, antagonism) to interpret experimental drug responses.
Molecular cloning and structural biology (Molecular Pharmacology; Lefkowitz) explain why different ligands have different efficacies (different receptor conformations) and enable modern drug discovery techniques.
Cell signalling frameworks (Alberts) place receptors into cellular contexts: how receptor activation leads to second messengers, cascades, feedback, and cellular outcomes.
Clinically relevant corollary: targeting receptor subtype, signalling bias, or regulatory machinery (e.g., GRKs/β-arrestins) can refine therapeutic action and reduce side effects.
Experimental methods & tips (practical)
Radioligand binding for Kd and Bmax; competition binding for Ki determination (Cheng–Prusoff relationship).
Functional assays: measure second messenger production (cAMP, IP3), ion flux, reporter gene activity for EC50/Emax.
Mutagenesis & chimeras to map binding pockets and coupling domains; heterologous expression for screening.
Use complementary data (binding + function + mutagenesis + structural data) for robust mechanistic conclusions.