G-Protein Coupled Receptors (GPCR)

• GPCRs are involved in many common diseases, including allergies, depression, blindness, diabetes, cardiovascular diseases
• Nearly 35% of all marketed drugs currently available work by targeting different GPCRS
• G proteins share a common feature: they can become activated and then, after a short time, can inactivate themselves
• G proteins serve as molecular binary switches with built-in timers
• Example: β-adrenergic receptor mediates effects of epinephrine

G-Protein Coupled Receptors (GPCR) - Overview

• Metabotropic receptors are transmembrane proteins composed of a single polypeptide chain
• GPCRs bind to a particular type of G protein (i.e.; G5, Gᵢ or Gq)
• GPCRs trigger different pathways: -cAMP signal pathway (Stimulatory - Gₛ and Inhibitory –Gᵢ) -Phosphatidylinositol (PIP2), DAG/IP3 pathway (Gq protein)
• GPCRs mediate most responses to signals from the external world, as well as signals from other cells
• There are about 900 types of GPCR, >350 for detecting hormones and other endogenous ligands and >500 olfactory and gustatory receptors

GPCR Mechanism - Components

• GPCRs are composed of: -Plasma membrane receptor (GPCR) with 7 transmembrane helical segments -G-protein (Gₛ, Gᵢ Gq...)
• Effector enzyme in the plasma membrane generating an intracellular second messenger -Guanosine nucleotide binding protein (GTP/GDP) which activates the G protein α -subunit
• When stimulated by receptor activation the G protein exchanges bound GDP for GTP

G-Proteins

• G-Proteins are membrane associated heterotrimeric proteins that transmit the signal into the cell interior
• Consist of 3 different subunits (α, β, and y)
• Bind directly to the cytoplasmic domain of GPCRs
• The α subunit (Gα) is a GTPase and has a GDP bound in its inactive state
• Gα once activated (by the ligand/GPCR complex) exchanges GDP for GTP
• The activation causes the dissociation of the β/γ complex from the a subunit
• Both the GTP-bound Gα and the β/γ complex can interact with targets
• May stimulate or inhibit effector proteins/enzymes -Ex: Enzymes and ion channels which transmit the signal onward
• Gα intrinsic GTPase hydrolyzes GTP to GDP and becomes inactive after a certain time (build in timer)

G-Proteins – Molecular switches

• The intrinsic GTPase activity of G proteins is increased by GTPase Activator Proteins (GAPs)
• GAPs strongly stimulate GTPase activity, causing rapid inactivation of the G protein
• Ga GTPase + GAP activities determine how long the switch remains 'on'

GPCR (Gₛ) Message Transduction - cAMP pathway

• Adenylate cyclase is adenylyl cyclase
• Ligand binds to the receptor, and G protein is activated
• Activated receptor stimulates adenylate cyclase
• Adenylate cyclase converts ATP to cAMP
• cAMP goes on to activate other proteins that alter cellular function
• The most important second messengers: Diacylglycerol (DAG), Inositol 1,4,5- Trisphosphate (IP3), Cyclic Adenosine Monophosphate (CAMP) and Calcium (Ca2+)

cAMP signaling pathway

• The binding of a signaling molecule activates GPCR that causes the G protein to bind GTP, promoting dissociation of the subunit from the β/γ dimer
• The binding of the α subunit to adenylyl cyclase promotes the synthesis of CAMP from ATP
• CAMP binds to the regulatory subunits of PKA, which releases the catalytic subunits
• The catalytic subunits of PKA phosphorylate specific cellular proteins and thereby cause a cellular response

cAMP - Second messenger effect

• Epinephrin binds to adrenergic receptors and exerts its downstream effect through the activation of adenylyl cyclase (AC) which increases [cAMP]
• cAMP allosterically activates PKA (PKA has catalytic and regulatory subunits)
• PKA catalyzes the phosphorylation of other proteins: E.g., Glycogen phosphorylase → mobilization of glycogen stores in liver and muscle in preparation for the need for energy
• Different proteins can be controlled by PKA because they all share an AA sequence that marks them for regulation by PKA (regulatory site)
• cAMP is ultimately reduced by cAMP phosphodiesterase (PDE)

Message Transduction – cAMP pathway

Steps for cAMP pathway from the receptor to response include:

• Binding of ligand to receptors causes conformational change that affects interaction with Gs
• Gs exchanges GDP for GTP and is activated
• The Gs α-subunit moves to Adenylyl Cyclase (AC) and activates it
• Adenylyl cyclase catalyzes the formation of cAMP
• cAMP activates Phosphokinase A (PKA)
• Phosphorylation of cellular proteins by PKA causes a cellular response
• Gs α is a GTPase that turns itself off by converting its bound GTP to GDP
• Binding of hormone activates GPCR to Gs
• Each active Gsa stimulates synthesis of many cAMP
• Each PKA phosphorylates many target enzymes
• Net effect of the cascade: the amplification of the hormone signal by several orders of magnitude

Stimulatory And Inhibitory G Proteins

• Some hormones inhibit AC, lowering [cAMP], and suppressing protein phosphorylation
• Example: somatostatin, adenosine, PGE1 binding to its receptor activates inhibitory G protein (Gi)
• Caffeine binds to Adenosine receptors
• Gi and Gs are structurally homologous, but have opposite effects

Signal Modulation and Termination

• Signal transducing systems must be able to turn off after the stimulus has ended
• Several mechanisms cause termination of the GPCR pathway
• Most systems adapt to the continued presence of the signal by becoming less sensitive in a process called desensitization: -Low concentrations of epinephrine in the blood cause its dissociation from its receptor and pathway inactivation -Intrinsic GTPase activity of G protein causes hydrolysis of bound GTP to GDP, causing return of Gα to bind with β and y subunits and consequently deactivation of the pathway
• GAPs GTPase: (activator proteins) speed up signal termination -cAMP production, and active PKA -Desensitization via receptor sequestration

Desensitization

• Process occurs in continued presence of epinephrine
• B-adrenergic receptor kinase (BARK) is drawn to receptor and phosphorylates it, creating a binding site for β-arrestin (β-arr)
• Binding of (β-arr) stops interaction between receptor and G protein
• Receptor removal is initiated through receptor sequestration: removing receptors from the membrane and taking them into intracellular vesicles through endocytosis
• Receptors dephosphorylate in endocytic vesicles, which causes the β-arr dissociates, and then receptors can return to membrane and re-sensitize the system to epinephrine

Signal Termination

• Desensitization reduces the cellular response even while the signal continues
• Five ways target cells can become desensitized to a signal molecule: -Receptor sequestration -Receptor down-regulation -Receptor inactivation -Inactivation of signaling protein -Production of inhibitory protein

Adrenergic Receptors

• Beta-1 adrenergic receptors are predominantly found in the heart -Epinephrine increases the heart rate and contractility (which helps pump out more blood) -Pathology/condition can cause tachyarrhythmias/high blood pressure
• Beta-2 adrenergic receptors are primarily found on smooth muscle cells, such as in the airways and lungs -Epinephrine causes bronchodilation (facilitating pulmonary ventilation - oxygen delivery to cells)
• Pathology/condition can cause narrowing of airways (Asthma)