G Protein-Coupled Receptors (GPCRs)

• G protein-coupled receptors (GPCRs) are also known as 7-transmembrane receptors
• Lehninger 8e is a major source of information on GPCRs

G-Protein Coupled Receptor (GPCR) Characteristics

• Contains 7-transmembrane α-helices
• Associates with heterotrimeric G-protein
• Heterotrimeric G-protein is a guanosine nucleotide-binding protein
• GDP and GTP are examples of guanosine nucleotides
• GDP is Guanosine-5'-diphosphate
• GTP is Guanosine-5'-triphosphate
• Ligand binding causes a receptor conformation change, altering the G-protein
• Over 30% of drugs target GPCRs
• The genome contains 800-900 GPCRs, which include some olfactory and taste receptors

Heterotrimeric G proteins

• These contain an α-subunit and a βγ-dimer
• The α-subunit binds nucleotide
• GDP bound to the α-subunit means the G protein is inactive
• GTP bound to the α-subunit means the G protein is active
• The α-subunit acts as a molecular switch to transduce signals
• It possesses inherent GTPase activity

Types of G-protein Coupled Receptors

• Gai inhibits adenylate cyclase, resulting in inhibition of cAMP and activation of phospholipases; this leads to cell motility
• Gas increases cAMP levels, resulting in cell growth and motility
• Gaq increases DAG and IP3 levels, resulting in cell proliferation
• Ga12 activates Rho, resulting in cancer progression and metastasis

β-adrenergic Receptor Signaling

• This is activated by Epinephrine (adrenaline), which is a ligand
• Isoproterenol is an agonist that also activates β-adrenergic receptor signaling
• Propranolol is an antagonist that inhibits β-adrenergic receptor signaling
• The β-adrenergic receptor signal transduction cascade uses Gα

β-adrenergic Cascade

• Epinephrine binds to its specific receptor, the β-adrenergic receptor
• Hormone-receptor complex causes the GDP bound to Gαs to be replaced by GTP, activating Gαs
• Activated Gαs separates and moves to adenylyl cyclase, activating it
• Adenylyl cyclase catalyzes the formation of cAMP
• cAMP activates protein kinase A (PKA)
• Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine
• cAMP is degraded reversing the activation of PKA

Adenyl Cyclase and cyclic AMP (cAMP)

• Adenyl cyclase acts on ATP to generate cyclic AMP (cAMP)
• cAMP is a second messenger
• Adenyl cyclase acts on ATP to produce cyclic AMP (cAMP) and PPi
• Cyclic nucleotide phosphodiesterase catalyses the breakdown of cAMP to Adenosine 5'-monophosphate (AMP)

Protein Kinase A (PKA)

• Inactive PKA contains 2 regulatory (R) subunits and 2 catalytic (C) subunits (R2C2)
• The autoinhibitory domain of the R subunit occupies the active site on the C subunit
• cAMP binds to the regulatory subunits, leading to a conformational change that releases 2 active C subunits
• PKA can phosphorylate several proteins in the cytosol and move to the nucleus

Epinephrine-Triggered Cascade

• A small number of molecules can result in a substantial intracellular signal due to amplification at several steps
• The numbers involved are likely underestimated

GTPase Activity Regulation

• Guanosine nucleotide exchange factors (GEFs) facilitate GDP/GTP exchange to activate G-proteins
• Excess GTP in the cellular environment is always available to bind to G-proteins
• GTPase activator proteins (GAPs) increase GTPase activity to inactivate G-proteins by 100,000x

The G-Protein Switch

• When GTP is bound, switch I and II are exposed and able to interact with targets
• The terminal phosphate hydrogen bonds with key residues
• When GTP is hydrolyzed, the protein relaxes, and the switch regions are buried

GPCR Signaling

• An animation illustrates GPCR signaling
• The alpha subunit and the beta-gamma dimer of the G-protein complex are indicated
• Di-phosphate and Guanosine molecules participate in the signaling
• The process involves a conformational change and the activation of adenyl cyclase, cAMP and Protein Kinase A

Adapter Proteins and AKAPs

• Adapter proteins, also known as docking proteins, are noncatalytic proteins that hold proteins together
• They enable proteins to perform their functions in concert
• AKAPs (A kinase anchoring proteins) are adaptor proteins that lack catalytic activity
• One side of AKAPs binds to a dimer of PKA's R subunits
• The other side can bind to a cell structure, such as the plasma membrane
• Different AKAPs bind different structures like actin filaments, ion channels, mitochondria, and the nucleus
• Different cells have different AKAPs

Termination of Response

• At every level of the signaling cascade, there is an "off switch"
• A "off switch" can include, ligand dissociation, metabolism/diffusion away, G-protein GTPase activity being turned off, etc.
• cAMP metabolism is regulated by cyclic nucleotide phosphodiesterases
• Protein phosphatases can remove added phosphates

Desensitization

• Decreasing the amount of receptor at the plasma membrane decreases the possibility of a response
• Decreasing even when the signal persists is desensitization
• Gsβγ recruits β-adrenergic protein kinase (BARK)
• PKA activates βARK
• βARK phosphorylates the receptor, leading to β-arrestin (βarr) recruitment
• β-arrestin causes endocytosis of the receptor, forming a negative feedback loop

G Protein and Arrestin Binding

• G protein and arrestin bind to the receptor in a mutually exclusive manner

Arrestin and the MAPK Pathway

• Binding of arrestin initiates a second pathway, the MAPK pathway

Signals Using cAMP as a Second Messenger

• Corticotropin, corticotropin-releasing hormone, dopamine, follicle-stimulating hormone, glucagon, histamine [H2], luteinizing hormone, and melanocyte-stimulating hormone
• Odorants, parathyroid hormone, prostaglandins E1/E2, serotonin [5-HT1, 5-HT4], somatostatin tastants (sweet and bitter), and thyroid-stimulating hormone

Gi-Associated GPCRs

• These inhibit adenylate cyclase
• Are structurally homologous to Gs

Gq Characteristics

• Part of a heterotrimeric G protein complex that binds to an associated receptor
• Activates phospholipase C (PLC) via direct association of Gq𝛼 to PLC
• PLC generates 2 second messengers: Ca2+ from the action of Inositol triphosphate (IP3), and Diacylglycerol (DAG)

PLC and Second Messengers

• PLC generates DAG and IP3 from PIP2

Gq Utilization

• GPCRs utilize Gq
• Hormones bind to specific receptors causing GDP-GTP exchange
• The now active Gq activates phospholipase C (PLC), which cleaves PIP2 into IP3 and Diacylglycerol
• Once released, IP3 binds to specific receptor-gated Ca2+ channels releasing Calcium 2+, activating protein kinase C

IP3 and Calcium Release

• IP3 causes release of intracellular calcium from the ER by opening the IP3-gated Ca2+ channel

Protein Kinase C

• The Protein Kinase C family is activated by DAG and Ca2+
• Classical PKC is activated by DAG and Ca2+, includes isoforms α, β1, β2, and γ
• Novel PKC is activated by DAG, includes isoforms δ, ε, η, and theta
• Atypical PKC is activated by phosphorylation, includes isoforms ζ and ι/λ

Classical PKC Activation

• In the inactive state, the PKC isozyme is in a "closed" conformation with the pseudosubstrate blocking the substrate binding region
• Following agonist-stimulated lipid hydrolysis, PKC is activated through a series of sequential activation steps
• PKC binds Ca2+ and translocates to membranes where binding of DAG occurs
• DAG binding promotes the activation and opening of the isozyme, which can now bind ATP and phosphorylate various substrates

PKC-Mediated Function

• Acetylcholine is a neurotransmitter released by neurons at the neuromuscular junction
• It activates muscle cells depending on where it is found
• The receptor in the smooth muscle of the respiratory system is the muscarinic acetylcholine receptor (M1 or M3)
• M1 or M3 are Gq-associated GPCRs that activate PKC leading to muscle constriction
• Sarin interferes with the degradation of Acetylcholine, causing muscles to stay constricted and blocking breathing
• This leads to death by asphyxiation
• Atropine, from the deadly Nightshade plant, blocks the action of Acetylcholine by competitively blocking binding

PKC and Muscle Contraction

• PKC is involved with regulating muscle contraction through Actomyosin Contractility

Muscle Regulation

• Myosin light chain phosphorylation by Myosin Light Chain Kinase (MLCK) promotes binding to actin to promote contraction
• Myosin light chain phosphatase (MLCP) removes phosphate to promote relaxation
• CPI-17 inhibits MLCP, keeping the phosphate on
• PKC phosphorylates and activates CPI-17

Signals Acting Through DAG, IP3, and Ca2+

• Muscarinic Acetylcholine (M1), 𝛼1-Adrenergic agonists, Angiogenin, Angiotensin II, ATP , Auxin, Gastrin-releasing peptide, Glutamate, Gonadotropin-releasing hormone
• Histamine , Light, Oxytocin, Platelet-derived growth factor (PDGF), Serotonin , Thyrotropin-releasing hormone, Vasopressin

G12/13 Overview

• G12/13 are activated by G-protein coupled receptors and regulate certain processes
• G12/13 promotes Chemorepellent Retraction

GPCRs Activating G12/13

• 30 GPCRs activate G12/13 heterotrimeric G proteins through direct or indirect detection

GPCR Coupling

• Some GPCRs can couple to multiple types of G proteins
• Crosstalk exists between the pathways
• For example, G𝛼12 is phosphorylated by PKC𝛼, 𝛿, 𝜀, 𝜁 to block the interaction of G𝛼12 with G𝛽𝛾

GPCR Complexity

• GPCRs exist as homo and heterodimers
• Dimerization increases the diversity of signaling responses

G Protein Function

• Different G proteins work together to form logic gates

G12/13 Regulation

• Activation of RH-RhoGEFs
• p115RhoGEF, PDZ-RhoGEF/GTRAP48, and leukemia-associated RhoGEF (LARG)
• Activated by binding to G12/13 via their RH domain
• RhoGEFs activate RhoA

RhoA

• Small molecular weight GTPase (single subunit)
• Binds GTP to be activated, GDP to inactivate
• Regulated by GEFs and GAPs just like heterotrimeric G proteins

RhoA Function

• Activation of the NF-𝜅B transcription factor downstream of RhoA
• Increases expression of genes that promote cell motility and proliferation
• LARG has been found to be mutated in human cancers

RhoA and cell morphology

• RhoA regulates cell motility and morphology via the assembly of contractive actin and myosin filaments in cells
• Also promotes cell contraction by contracting the trailing edge of the cell

Rhodopsin

• Rhodopsin is a GPCR in the rod cells of the eye
• 100-125 million rod cells contribute to being able to see in low light
• Cone cells are responsible for seeing color

Rhodopsin cascade

• Absorption of light converts 11-cis-retinal to all-trans-retinal, which activates rhodopsin
• Activated rhodopsin catalyzes replacement of GDP with GTP on transducin (T) which dissociates into T𝛼-GTP and T𝛽𝛾
• T𝛼-GTP activates cGMP phosphodiesterase (PDE) by binding and removing its inhibitory subunit (I)
• Active PDE reduces the concentration of cGMP so that cation channels close and prevent the influx of Na and Ca
• Membrane is hyperpolarized which causes a signal to be passed to the brain

Phosphodiesterase - GMP Reactions

• Cyclic GMP (cGMP) is reduced to GMP by phosphodiesterase (PDE)
• GMP is the result of action of phosphodiesterase on cGMP.

Cyclic GMP

• Decreased cAMP closes channels

Signal Termination

• Signal needs to be turned off to be able to dectect the next signal
• Arrestin disassociates, rhodopsin is dephosphorylated and the cell is ready for another signal