Rucete ✏ Lehninger Principles of Biochemistry In a Nutshell
12.2 G Protein–Coupled Receptors and Second Messengers
This chapter explains how G protein–coupled receptors (GPCRs) detect extracellular signals and convert them into intracellular responses through G proteins, second messengers such as cAMP, protein kinase A, receptor desensitization, and signaling integration. GPCR systems are among the most important signaling mechanisms in human biology and medicine.
What Are GPCRs?
• G protein–coupled receptors (GPCRs) are membrane receptors that transmit signals through guanosine nucleotide-binding proteins called G proteins.
• Three essential components define GPCR signaling:
• A plasma membrane receptor with seven transmembrane α-helices.
• A G protein that switches between inactive GDP-bound and active GTP-bound states.
• An effector enzyme or ion channel regulated by the activated G protein.
General Mechanism of GPCR Signaling
• An extracellular ligand such as a hormone or neurotransmitter acts as the first messenger.
• Ligand binding changes receptor conformation allosterically.
• The activated receptor interacts with a nearby G protein.
• GDP on the G protein is exchanged for GTP.
• The activated G protein regulates an effector enzyme or ion channel.
• The effector changes levels of intracellular second messengers such as cAMP or Ca²⁺.
• Second messengers activate downstream targets, often protein kinases.
Importance of GPCRs in Humans
• The human genome encodes just over 800 GPCRs.
• About 350 detect hormones, growth factors, and endogenous ligands.
• Up to 500 function in smell and taste.
• GPCRs are the largest protein superfamily in the human genome.
• They are involved in allergies, depression, blindness, diabetes, and cardiovascular disease.
• GPCR mutations occur in about 20% of cancers.
• More than one-third of marketed drugs target GPCRs.
Example: Beta Blockers
• β-adrenergic receptors respond to epinephrine.
• Beta-blocker drugs inhibit these receptors.
• They are used for hypertension, arrhythmia, glaucoma, anxiety, and migraine.
Orphan Receptors
• More than 100 human GPCRs are orphan receptors.
• Their natural ligands remain unknown.
• Their biological roles are still being studied.
The β-Adrenergic Receptor System
• The β-adrenergic receptor is a classic model GPCR.
• It mediates the effects of epinephrine (adrenaline).
• Epinephrine is released from adrenal glands during stress.
• It prepares the body for fight-or-flight responses.
• Target tissues include muscle, liver, and adipose tissue.
Adrenergic Receptor Types
• There are four general adrenergic receptor classes: α₁, α₂, β₁, and β₂.
• They differ in ligand affinity and downstream response.
• Different tissues express different receptor subtypes.
• This allows one hormone to create multiple physiological effects.
Agonists and Antagonists
• Agonists bind receptors and activate them.
• Antagonists bind receptors but block activation.
• Synthetic compounds may bind receptors more strongly than natural ligands.
Structure of GPCRs
• GPCRs are integral membrane proteins.
• They contain seven hydrophobic membrane-spanning helices.
• Therefore they are also called seven-transmembrane (7tm) or heptahelical receptors.
Heterotrimeric G Proteins
• GPCRs signal through heterotrimeric G proteins composed of α, β, and γ subunits.
• GDP or GTP binds the α subunit.
• GDP-bound form is inactive.
• GTP-bound form is active.
Receptor as a GEF
• Activated GPCRs function as guanine nucleotide exchange factors (GEFs).
• They promote release of GDP from Gα.
• Cytosolic GTP then binds Gα.
• This activates the G protein.
Gs and Adenylyl Cyclase
• In β-adrenergic signaling, the relevant G protein is stimulatory G protein (Gs).
• Activated Gαs-GTP separates from βγ.
• Gαs moves laterally in the membrane and binds adenylyl cyclase.
• This activates adenylyl cyclase.
Adenylyl Cyclase
• Adenylyl cyclase is an integral membrane enzyme.
• Its catalytic site faces the cytosol.
• It converts ATP into cyclic AMP (cAMP).
• cAMP acts as a second messenger.
• Cytosolic cAMP concentration rises after receptor stimulation.
Self-Limiting G Protein Switch
• Gα subunits possess intrinsic GTPase activity.
• GTP is hydrolyzed to GDP.
• Gα returns to the inactive state.
• It dissociates from adenylyl cyclase.
• GαGDP reassociates with βγ to reform inactive heterotrimeric G protein.
GTPase Activator Proteins
• GTPase activator proteins (GAPs) accelerate GTP hydrolysis.
• They shorten signaling duration.
• GAP activity can also be regulated.
cAMP Activates Protein Kinase A (PKA)
• cAMP activates cAMP-dependent protein kinase, also called protein kinase A (PKA).
• PKA phosphorylates Ser or Thr residues on target proteins.
• This alters enzyme activity and cell behavior.
Inactive Structure of PKA
• Inactive PKA contains:
• Two catalytic (C) subunits.
• Two regulatory (R) subunits.
• The R subunits contain autoinhibitory domains that block catalytic active sites.
Activation of PKA
• cAMP binds the regulatory subunits.
• Regulatory subunits change conformation.
• Autoinhibitory domains move away.
• Catalytic subunits are released.
• Free catalytic subunits become active kinases.
Common Kinase Principle
• Many protein kinases are activated by displacement of inhibitory domains.
• PKA serves as a prototype for many kinase systems.
PKA Substrate Recognition
• PKA phosphorylates proteins containing specific consensus sequences near Ser or Thr residues.
• This sequence selectivity allows targeted regulation of many proteins.
Downstream Effects of Epinephrine
• PKA activates phosphorylase b kinase.
• Phosphorylase b kinase activates glycogen phosphorylase.
• Glycogen breakdown increases.
• Glucose becomes rapidly available for energy use.
Signal Amplification
• GPCR signaling strongly amplifies weak signals.
• One hormone-bound receptor can activate many G proteins.
• One adenylyl cyclase makes many cAMP molecules.
• One PKA phosphorylates many targets.
• This cascade can create huge cellular responses from tiny hormone concentrations.
Rapid Response
• Intracellular changes can occur within fractions of a second.
Termination of β-Adrenergic Signaling
• Signaling must stop after stimulus removal.
• Several mechanisms terminate the response.
1. Ligand Dissociation
• When epinephrine concentration falls below receptor affinity threshold, it dissociates.
• The receptor returns to inactive form.
2. GTP Hydrolysis
• Gα hydrolyzes bound GTP to GDP.
• Adenylyl cyclase stimulation stops.
3. cAMP Breakdown
• Cyclic nucleotide phosphodiesterase hydrolyzes cAMP to 5′-AMP.
• 5′-AMP is not a second messenger.
4. Dephosphorylation of Targets
• Phosphoprotein phosphatases remove phosphate groups from proteins.
• Tyr, Ser, or Thr phosphorylation states are reversed.
• Human genome encodes about 190 phosphatases.
• PP1 alone dephosphorylates many substrates.
Desensitization during Continuous Stimulation
• If epinephrine remains present, receptor responsiveness decreases even before ligand disappears.
• This is called desensitization.
β-Adrenergic Receptor Kinase (ARK / GRK)
• β-adrenergic receptor kinase phosphorylates Ser residues near the receptor C-terminus.
• It is recruited to the membrane by Gβγ.
• ARK belongs to the GPCR kinase (GRK) family.
• Humans encode seven GRKs.
Arrestin Binding
• Phosphorylated receptor binds β-arrestin.
• Arrestin blocks receptor interaction with G proteins.
• This stops further signaling through G proteins.
Endocytosis of Receptors
• Arrestin also recruits clathrin and vesicle-forming proteins.
• Receptors are internalized into endosomes.
• Internalized receptors cannot bind extracellular epinephrine.
Resensitization
• Receptors are later dephosphorylated.
• They return to the plasma membrane.
• Full sensitivity is restored.
Arrestin as a Signaling Protein
• Arrestin does more than shut off receptors.
• Receptor-arrestin complexes can activate MAPK signaling pathways.
• Thus one GPCR can initiate two divergent pathways:
• G protein pathway
• Arrestin pathway
Biased Agonism
• Some ligands preferentially activate G-protein signaling.
• Others favor arrestin signaling.
• This is important in drug design.
• Example: ideal opioid drugs would preserve pain relief while minimizing addiction pathways.
Many Signals Use cAMP
• cAMP is used by numerous hormones and signals, including:
• Glucagon
• ACTH
• TSH
• LH
• FSH
• Parathyroid hormone
• Histamine
• Serotonin (certain receptor types)
• Many odorants and tastants
Examples of cAMP Actions
• Glucagon in adipocytes raises cAMP and mobilizes stored fats.
• ACTH in adrenal cortex raises cAMP and stimulates cortisol synthesis.
• In many cells, catalytic PKA subunits enter the nucleus and phosphorylate CREB.
• CREB changes transcription of cAMP-responsive genes.
Inhibitory Gi Proteins
• Some receptors activate inhibitory G proteins (Gi).
• Gi inhibits adenylyl cyclase.
• cAMP decreases.
• PKA signaling falls.
Examples of Gi Signaling
• Somatostatin inhibits glucagon secretion through Gi pathways.
• In adipose tissue, prostaglandins can reduce cAMP and slow fat mobilization.
Same Ligand, Different Effect
• The same extracellular signal may act differently in different tissues depending on:
• Receptor subtype present
• Which G protein is coupled
• Which downstream target proteins are expressed
Signal Integration
• Cells sum pathways that raise cAMP and lower cAMP.
• Final cAMP level determines response strength.
AKAPs (A Kinase Anchoring Proteins)
• AKAPs are multivalent adaptor proteins.
• They bind regulatory subunits of PKA.
• They anchor PKA to specific cell locations such as:
• Microtubules
• Actin filaments
• Ion channels
• Mitochondria
• Nucleus
Importance of AKAPs
• Different cells contain different AKAP sets.
• Therefore cAMP signaling can regulate different local targets in different tissues.
• Some AKAPs also bind adenylyl cyclase, phosphodiesterase, or phosphatases.
• This creates highly localized and brief signaling microdomains.
FRET and Live-Cell Signaling Analysis
• Fluorescence resonance energy transfer (FRET) can detect protein interactions in living cells.
• It measures energy transfer between nearby fluorescent probes.
• FRET has been used to monitor cAMP concentration and PKA activation in real time.
In a Nutshell
GPCRs are seven-transmembrane receptors that convert extracellular signals into intracellular responses through heterotrimeric G proteins and second messengers such as cAMP. In the β-adrenergic pathway, epinephrine activates Gs, adenylyl cyclase, cAMP, and PKA, leading to rapid signal amplification and metabolic change. Signaling is controlled by GTP hydrolysis, phosphodiesterases, phosphatases, GRKs, arrestins, receptor internalization, and localized AKAP complexes.