W28. G-protein coupled receptors

Receptor Signalling

• Agonist binding to its receptor leads to the formation of a drug-receptor (D-R) complex
• The D-R complex then undergoes a conformational change
• This conformational change triggers a chain of biochemical processes
• Ultimately, this biochemical cascaderesults in a biological response
• This process is termed 'signal transduction' or 'receptor signalling'
• The biochemical events triggered inside the cell forms ‘the signalling cascade’ or ‘signal transduction pathway’
• Receptor signalling involves signal reception, signal transduction, and a cellular response

Signal Reception

• An agonist drug binds to and activates a specific receptor on or inside the target cell

Signal Transduction

• The drug-receptor complex activates a series of relay proteins, leading to the production of second messengers in the cell

Cellular Response

• Eventually, a cellular or biological response to the original drug-binding signal is triggered

Signal Transduction Pathways

• There are four major signal transduction pathways
• Activation of receptor-ion channels (ligand-gated receptors)
• Activation of second messenger pathways via G-protein-coupled receptors
• Activation of enzyme-linked receptors (e.g., tyrosine kinase-linked receptors)
• Direct activation of gene transcription via intracellular receptors

Receptor Superfamilies

• A receptor superfamily is a group of receptors sharing a similar basic molecular structure and utilizing the same signal transduction pathway
• Four major receptor superfamilies exist

Four Major Receptor Superfamilies

• Ligand-gated/ion channel-linked receptors
• G-protein-coupled receptors
• Enzyme-linked/kinase-linked receptors
• Intracellular/nuclear receptors

G-Protein-Coupled Receptors (GPCRs)

• GPCRs, also known as metabotropic receptors, form a large and diverse superfamily of integral membrane proteins
• These convert extracellular signals into intracellular responses
• GPCRs represent the largest receptor superfamily in humans, with approximately 800 members
• They transduce a variety of extracellular signals, regulating nearly every aspect of physiology
• These receptors mediate responses to hormones, neurotransmitters, growth factors, vision, olfaction, and taste signals
• GPCRs are the targets for around 40% of drugs on the pharmaceutical market

Key Features & Characteristics of GPCRs

• All share a common structural motif of seven transmembrane α-helices (7-TM)
• They couple to and activate cytoplasmic heterotrimeric G-proteins upon agonist binding
• This coupling modulates downstream effector proteins, resulting in a biological response
• GPCRs also couple to cytoplasmic adaptor proteins called β-arrestins, leading to receptor desensitization, internalization, or activation of downstream effector proteins

Structure of GPCRs

• A GPCR possesses a single polypeptide chain, comprising three key regions
• The extracellular region includes the N-terminus and three extracellular loops (ECL1-ECL3), modulating ligand access to the binding site
• The TM region features seven transmembrane α-helices (7-TM), labeled TM1-TM7, forming the structural core that binds ligands and transduces information to intracellular regions
• The intracellular region contains three intracellular loops (ICL1-ICL3), a short intracellular α-helix (H8), and the C-terminus, interfacing with cytosolic signaling proteins like G-proteins

GPCR Signaling via Heterotrimeric G-Proteins

• The defining characteristic of GPCRs is their interaction with heterotrimeric GTP-binding proteins, or G-proteins
• Heterotrimeric G-proteins are crucial in the signal transduction pathways initiated by GPCR activation
• They reside at the inner leaflet of the plasma membrane, relaying signals from cell-surface GPCRs to downstream intracellular effector proteins
• G-proteins act as molecular binary switches, translating agonist-GPCR binding into modulation of activity of downstream intracellular effector proteins to produce a biological response

Heterotrimeric G-Proteins

• Composed of three different protein subunits: α, β, and γ
• Functionally composed of two units: an α subunit (Gα) and a tightly associated βγ complex
• Gα and Gγ subunits have lipid extensions that bind and tether the G-protein complex to the plasma membrane
• The Gα subunit has a guanine nucleotide-binding site, occupied by GDP in the inactive, resting state
• To date, 21 Gα, 5 Gβ, and 12 Gγ subunits/isoforms are apparent in the human genome, creating multiple permutations of distinct heterotrimeric complexes

Heterotrimeric G-Protein States

• The Gα subunit contains a Ras-like domain and an α-helical domain
• A nucleotide-binding pocket resides between the two domains
• The Ras-like domain exhibits GTPase activity, hydrolyzing GTP to GDP, and provides binding sites for the Gβγ subunits

Gα Subunit

• The N-terminus is myristoylated or palmitoylated
• This modification anchors the G-protein to the plasma membrane

Signaling via Heterotrimeric G-Proteins

• Agonist-bound GPCR induces a conformational change, prompting the exchange of GDP for GTP on the Gα subunit and dissociation of the Gα-GTP subunit from the Gβγ dimer
• Gα-GTP and the freed Gβγ each interact with unique downstream effector proteins, regulating their activity and leading to a biological response
• The activated G-protein eventually reverts to its inactive resting state
• GTPase activity in the Gα subunit hydrolyzes bound GTP to GDP
• Regulators of G-protein signaling (RGS) proteins or GTPase-accelerating proteins (GAPs) accelerate GTP hydrolysis to GDP
• Gα-GDP then re-assembles with the Gβγ dimer, reforming the inactive G-protein

Diversity of GPCR Signaling Mechanisms

• G proteins are classified per their Gα subunits
• Gα proteins are grouped into 4 families based on peptide sequence and functional similarities: Gαs, Gαi, Gαq/11, and Gα12/13 protein families
• Each Gα family can relay GPCR signals to multiple downstream effectors, triggering diverse signaling pathways

GPCR Signalling Mechanisms - Gα Proteins

• Gαs family stimulates adenylate cyclase, increasing cAMP levels
• Gαi family inhibits adenylate cyclase, decreasing cAMP levels
• Gαq/11 family stimulates phospholipase C-β, increasing IP3 and DAG levels
• Gα12/13 family activates the Rho family of GTPases

Gᴀ-Mediated Signaling Pathways & Second Messengers

• Most Gα proteins mediate GPCR signalling by regulating the levels of intracellular regulatory molecules, called second messengers
• The second messengers regulate the activity of multiple downstream effector proteins, leading to a biological response
• Key second messengers include cAMP, IP3, and Ca2+

cAMP Signaling

• Gαs-GTP activates adenylyl cyclase (AC), increasing cAMP levels
• Gαi/o-GTP inhibits adenylyl cyclase (AC), decreasing cAMP levels

cAMP & Adenylyl Cyclase

• Gαs-GTP stimulates the activation of adenylyl cyclase (AC), a membrane-bound enzyme
• Adenylyl cyclase (AC) converts ATP to cAMP
• Increased cAMP levels activate protein kinase A (PKA)
• cAMP can also modulate the activity of several guanine exchange factors (GEFs) and ion channels
• Galpha i/o-GTP inhibits the activity of AC and lowers cellular cAMP levels, decreasing PKA activation

IP3/Ca2+ Signaling Pathway

• Gαq/11-GTP activates phospholipase C-β (PLCβ)
• Gαq/11-GTP activates phospholipase C-β (PLCβ), increasing IP3 & DAG levels

IP3, DAG & Phospholipids

• Phosphatidylinositol-bisphosphate (PIP2) is a membrane phospholipid
• Cleaved by activated phospholipase C-β (PLCβ) to generate second messengers: inositol triphosphate (IP3) and diacylglycerol (DAG)
• IP3 diffuses through the cytosol, releasing Ca2+ from the ER
• DAG stays in the membrane, activating protein kinase C

GPCR Desensitization and Intracellular Trafficking

• Temporal and spatial signaling of activated GPCRs is governed by desensitization and internalization
• Involves sequential action of two major intracellular actors: G-protein-coupled receptor kinases (GRKs) & β-arrestins (cytoplasmic adaptor proteins)
• GRK docks onto activated GPCR, phosphorylating serine and threonine residues to inhibit G-protein activation (homologous desensitization)
• PKA, PKC, and other S/T kinases can phosphorylate inactive GPCRs, leading to heterologous desensitization
• Next, β-arrestins bind to phosphorylated GPCRs, sterically hindering GPCR-G protein coupling to inhibit further activation of G-protein signaling
• b-arrestins then couple the phosphorylated GPCRs to clathrin-coated pits, facilitating receptor internalization
• internalised GPCRs traffic to endosomes with one of 3 fates
  • They may be dephosphorylated by phosphatases
  • Recycle to the cell surface for future signaling
  • May be sorted to lysosomes for degradation
• They may activate β-arrestin-dependent and G-protein-independent GPCR signaling cascades

Diversity of GPCR Signalling Mechanisms – Signalling via β-Arrestins

• Rapid attenuation or desensitization prevents uncontrolled signaling following GPCR activation
• Desensitization is initiated by phosphorylation of the receptor by GPCR kinases, followed by uncoupling of GPCR-G protein interactions mediated by β-arrestins
• In addition to terminating G protein signalling, β-arrestins promote GPCR signalling by internalizing the receptor and acting as a molecular scaffold to recruit signaling proteins
• ß-arrestins initiate G protein-independent GPCR signalling cascades

Neurotransmitters and GPCRs

• GPCRs mediate the actions of key chemical messengers, including roles for adrenergic actions, acetylcholine actions, dopamine actions, and serotonin.

Adrenergic Actions

• Can involve Norepinephrine and Epinephrine

Acetylcholine Actions

• Can involve Acetylcholine and Carbachol

Dopamine Actions

• Dopamine can activate the D1/D5 or the D2/D3/D4 complexes

Serotonin Actions

• Serotonin activates the 5-HT subtypes