Signalling via G-protein Coupled Receptors

HUBS2206 Human Biochemistry and Cell Biology Lecture 28: Signalling via G-protein Coupled Receptors

Learning Targets

• What is a GPCR?
• How do GPCRs activate a signalling cascade? How is the signalling cascade terminated?
• What are second messengers?
  • Need to know types and how they are generated
• What is signal amplification?
  • Example: Adrenaline and cAMP signalling
• The diversity of GPCR signalling:
  • GPCR, G-protein and second messenger diversity
• Key concept: Signal amplification via GPCR
  *Note: Need to know all the text and examples, except what is marked “for reference”

Case Study: Fight or Flight!

• How do you respond to sudden short-term stress?
• Immediate short-term response to crises
  • Adrenaline (epinephrine)
  • Noradrenaline

Alarm Phase

• Mobilization of glucose reserves
• Changes in circulation
• Increased heart and respiratory rates
• Increased energy use by all cells

Adrenaline (Epinephrine)

• How does adrenaline work?
• Adrenaline binds to GPCRs.
• Mobilisation of glucose reserves in the liver and muscle.
• Increased heart muscle contraction.
• Adrenaline activates tissue-specific GPCRs leading to production of cAMP.
• Need a fast and strong cell response.

G-Protein Coupled Receptors (GPCRs)

• Largest family of cell receptors.
  • Over 800 GPCRs in humans. Largest family of membrane proteins in the human genome!
• Activated by a large variety of signalling molecules.
  • Proteins, neurotransmitters, peptide hormones, ions, vitamins, metabolites, photons, fatty acids, all molecules that we can smell or taste.
• Involved in physiological processes in most tissues in the body.
• Single polypeptide chain spans back and forth across the membrane seven times.
• Binding of ligand to receptor causes conformational change in receptor.
• This promotes binding to a “G-protein” = heterotrimeric GTP-binding protein (not small GTPase like Ras).
• GPCRs are “seven-pass” transmembrane domain receptors.

Heterotrimeric G-Proteins

• Composed of three subunits: α, β, and γ.
  • α and γ subunits are attached to the membrane.
• In unstimulated (resting) state, α-subunit has GDP bound, thus is inactive.
• Upon receptor activation, exchange of GDP with GTP activates the α-subunit (which dissociates from Gβ and Gγ).
• Hydrolysis of GTP returns everything to resting stage.
• GTP binding acts as a molecular switch.

Effectors of Activated G-Proteins

• Enzymes: Most common
  • E.g., adenylate cyclase becomes activated.
  • In less common cases, effectors can also become inhibited, e.g., adenylate cyclase.
• Ion channels: Affects ion flux into cells.
• Tubulin: Affects microtubule dynamics.

Signalling Via GPCR and Second Messengers

• Principles and important examples
  • Generation of cAMP, IP3, DAG, calcium, cGMP, NO.

Key Concept

• Most often, activated Gα subunits associate with and activate effector enzymes resulting in generation of second messengers.
• Classical example:
  • Effector = adenylate cyclase
  • Second messenger = cAMP

What are Second Messengers in Signalling?

• Second messengers disseminate information received from cell-surface receptors.
• Elevated concentrations of second messengers promote their binding to specific cellular enzymes, altering their activity to relay downstream signals.
• Short-lived intracellular signalling molecules: Removal or degradation terminates the cellular response.

Four Major Classes of Second Messengers

1. Cyclic nucleotides: e.g., cAMP, cGMP
   • Signal within cell cytoplasm
2. Ions: e.g., Calcium ($Ca^{2+}$)
   • Ions signal within and between cellular compartments
3. Membrane lipid derivatives: Such as inositol triphosphate (IP3), diacylglycerol (DAG)
   • Lipid messengers signal within cell membranes
4. Gases (e.g., nitric oxide) and free radicals
   • Can signal throughout cell and even to neighbouring cells
     *Note: Some second messengers like calcium can also be produced in response to stimuli from within the cell.

cAMP = Second Messenger

• Adenylate cyclase catalyses the conversion of ATP to cyclic AMP (cAMP), which can bind to and activate protein kinase A by dissociating a regulatory subunit.
  *Note: cAMP can be rapidly broken down by enzymes, which leads to termination of signalling.

Example: Epinephrine-Stimulated cAMP Synthesis

Resting State:

• Inactive adenylyl cyclase and G-protein.

Stimulated State:

• Epinephrine binds to beta-adrenergic receptor.
• G-protein dissociation.
• Adenylyl cyclase converts ATP to cAMP.
  *Leads to increased heart rate, dilation of skeletal muscle blood vessels, breakdown of glycogen to glucose.

GPCR and Signal Amplification

• Hydrophilic signalling molecules often in low concentration.
• Activate relatively few receptors.
• Signal transduction can generate signal amplification.

Example: Adrenaline Signal Amplification

• Adrenaline causes breakdown of glycogen stores.
• One molecule of epinephrine binding to receptor $\longrightarrow$ 108 molecules of glycogen converted to glucose-1-phosphate $\longrightarrow$ glucose.

GPCR and Inositol Phospholipid Signalling

• Effector = phospholipase C
• Cleaves PIP2 to give IP3 and diacylglycerol (DAG) = second messengers.
• IP3 acts on $Ca^{2+}$ channel to release $Ca^{2+}$ = second messenger.
• Both DAG and $Ca^{2+}$ activate downstream protein kinases.
• Many GPCRs act through the breakdown of membrane phospholipids.

GPCR and Nitric Oxide (Gas)

• GPCR activation of endothelial cells lining blood vessel cause synthesis of nitric oxide (NO).
• NO rapidly diffuses across membranes and activates the soluble enzyme, guanylate cyclase (sGC), that converts GTP to cGMP, a second messenger.
• cGMP signalling leads to relaxation of smooth muscle cells that surround blood vessel, allowing vessel to dilate.
• Nitric oxide has short half-life (5-10 s).
• Nitroglycerine is used to treat patients with angina because it is converted to NO – relaxation of vessels reduces heart workload and oxygen requirement.
• Activation of cGMP, a second messenger.
  *NO = second messenger.

Specificity of Signalling Determined by Receptor and G-Protein Type

Many Water Soluble Hormones Activate GPCRs

*Note: Epinephrine: the same hormone can induce distinct signal transduction pathways depending on GPCR and G-protein types!

Conclusions

• GPCR signalling represents a major mechanism for signal transduction and activation or deactivation of pathways within the cell.
• A large part of efforts towards drug development today is focused on finding new drugs that affect the ability of ligands to bind to GPCRs either to inhibit or accelerate certain cellular processes.

GPCRs are Important Therapeutic Targets

• Target of ~30% of FDA approved drugs!
• Due to their physiological relevance in most body’s tissues

Targeting GPCRs: The Good… and the Bad

• ~50 GPCR peptide drugs have been approved to date
• Majority are agonists (activate receptors) targeting metabolism
• However, many more to target! (800 GPCRs in human)
• Big interest in cancer field as emerging anti-cancer drugs

HOWEVER

• GPCRs represent frequent off-targets for drugs
  • E.g., appetite suppressant drug fenfluramine (Fen-Phen) was popular to combat obesity
  • It increases the release of serotonin, a neurotransmitter that regulates mood, appetite, and other functions
  • Had to be withdrawn by FDA because it triggered serious cardiopulmonary side-effects (via activation of specific serotonin receptors)
• New compounds that target kinases and other non-GPCR molecular targets are typically profiled against large numbers of cloned GPCRs prior to clinical trials in humans!