G Proteins and GPCRs

G Proteins Activated by GPCRs

• Trimeric G proteins are distinct from small monomeric G proteins like Rabs.
• βγ subunits are inseparable and anchored to the plasma membrane.
• α subunits are divided into 4 major families and bind and hydrolyze GTP.

α Subunit Structure and Function

• GDP is bound to the inactive α-subunit in a deep pocket, restricting its escape.
• Two conserved residues within two distinct domains of the α subunit are switches 1 and 2.
• These switches make contact with the γ phosphate and pull in the switch regions, activating the G protein and breaking its contact with the βγ subunit.

G-Protein Activation and Dissociation

• GPCRs promote G-protein activation, opening the deep pocket where GDP is tightly bound.
• Binding of GTP to the α subunit causes dissociation from the βγ subunit.
• The α-GTP and βγ subunits can now regulate their effectors.

Regulation of Activity

• GTPase activity of α-GTP directly controls its activity and indirectly controls the activity of the βγ subunit.
• The α-GTP subunit reassociates with the βγ subunit once it has been hydrolyzed to α-GDP.

α Subunit Families and Functions

• The α_s subunit stimulates adenylyl cyclase.
• The α_i subunit inhibits adenylyl cyclase and is involved in the parasympathetic response to ACh.
• The α_q subunit stimulates phospholipase C.
• The α_12 subunit regulates the cytoskeleton.

βγ Subunit Functions

• The βγ subunit stimulates phospholipase C, inhibits Ca2+ channels, stimulates K+ channels, and stimulates P13K.

Toxins and Modifiers

• Cholera toxin covalently modifies α_s, blocking GTPase activity and leading to fluid loss in the gut.
• Pertussis toxin covalently modifies α_i, leading to uncoupling of the G-protein from GPCRs.

GPCR Structure

• All GPCRs possess a similar structure, featuring an extracellular N-terminal region and a cytosolic C-terminal region separated by 7 transmembrane domains (TMDs).
• The 7 TMDs are crucial for GPCR function, as they create a cleft when activated.

GPCR Activation

• When a GPCR is activated, a cleft opens between the cytosolic ends of TMDs 3, 5, 6, and 7.
• The cleft created by TMDs 3, 5, 6, and 7 serves as a binding site for the α-subunit of trimeric G proteins.
• The GPCR causes the release of GDP from the G-protein following binding.

Allosteric Proteins and Signaling Cascades

• Allosteric proteins transmit information from the plasma membrane (PM) via signaling cascades involving protein-protein and protein-small messenger interactions.

cAMP and Adenylyl Cyclases

• cAMP is an intracellular messenger produced from ATP by adenylyl cyclases.
• cAMP is inactivated by phosphodiesterases (PDEs).
• Protein kinase A (PKA) is the primary target of cAMP.

Adenylyl Cyclase (AC) Forms and Regulation

• There are 9 forms of adenylyl cyclase, all stimulated by α_s-GTP.
• Each form of AC responds differently to other intracellular signals, such as Ca2+.
• AC1 is stimulated by Ca2+ and responds optimally with Ca2+ and α_s-GTP, functioning as an AND gate.
• Other forms of AC function as NOT gates, demonstrating integrative behaviors.

Phosphodiesterases (PDEs) and Regulation

• PDEs are regulated and most are inhibited by caffeine and theophylline.
• Inhibition of PDEs increases cAMP levels, contributing to the treatment of asthma.

Therapeutic Applications

• Drugs that increase cAMP formation or reduce degradation can contribute to the treatment of asthma.
• Ventolin, which activates receptors in the airway, increases cAMP and relieves asthma.
• Cilostazol, which targets PDE3, is used to treat obstructed peripheral arteries.

Protein Kinase A (PKA)

• Consists of 2 regulatory (R) subunits, each binding 2 cAMP molecules, and 2 catalytic (C) subunits, which phosphorylate substrates
• R subunits have a pseudo-substrate domain that blocks the active site of the C subunit
• Binding of cAMP to R subunits causes them to fall apart, unblocking the active site of the C subunit, allowing phosphorylation of substrates
• Phosphorylation occurs on serine and threonine residues
• Phosphorylation is reversed by phosphatases

A-Kinase Anchoring Proteins (AKAPs)

• Scaffold proteins that associate with the R subunit of PKA
• Anchor PKA at different places within the cell
• Assemble related proteins with PKA, including targets of PKA

cGMP Signaling

• Similar to cAMP signaling
• Produced by guanylyl cyclases
• Degraded by phosphodiesterases (PDEs)
• Many actions mediated by protein kinase G

PDE5 and Viagra

• PDE5 selectively degrades cGMP
• Viagra prevents degradation of cGMP in blood vessels of the penis, causing them to dilate and accumulate blood

Phospholipase C (PLC) Signaling Pathway

• PIP2 is converted to DAG and IP3 by phospholipase C (PLC)
• DAG remains in the plasma membrane (PM) and activates protein kinase C (PKC)

DAG Metabolism

• DAG is converted to phosphatidate, stimulated by phosphorylation

IP3 Metabolism and Function

• IP3 is water-soluble and enters the cytosol, stimulating Ca2+ release from the endoplasmic reticulum (ER)
• IP3 is converted to inositol via IP1, stimulated by dephosphorylation and inhibited by lithium

Lithium's Effect on IP3 Metabolism

• Lithium blocks IP1, preventing the conversion to inositol, and is used to treat bipolar disorder by reducing signaling in over-active areas of the brain
• Lithium prevents PIP2 reformation, reducing signaling in over-active areas of the brain

Phosphatidate and Inositol Conversion

• Both phosphatidate and inositol are converted to PIP2

PLC Activation and Downstream Signaling

• PLC can be activated by G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs)
• Activated PLC produces DAGs, recruiting PKC to the PM and activating it, and IP3, releasing Ca2+ from the ER through IP3 receptors
• Ca2+ released by IP3 receptors regulates many intracellular activities, often through the highly conserved Ca2+ binding protein, calmodulin

IP3 Receptor and Ca2+ Signals

• IP3 receptor provides Ca2+ ions, which leads to the CICR (Calcium-Induced Calcium Release) mechanism.
• Increased stimulus intensity leads to more frequent Ca2+ waves.
• Ca2+ indicators developed by Tsein have revealed the spatial and temporal complexity of intracellular Ca2+ signals.

Characteristics of Ca2+ Signals

• Many Ca2+ signals are delivered as Ca2+ spikes.
• The frequency of Ca2+ spikes increases with stimulus intensity.
• Ca2+ spikes may protect cells from damaging effects of excessive increases in cytosolic Ca2+ concentrations.
• Ca2+ spikes allow for digital signalling, similar to action potentials.
• Spatially organised Ca2+ signals enable Ca2+ from different sources to be delivered to different targets.