Signalling via G-protein Coupled Receptors

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

Learning Targets

• GPCR Definition: Understanding what a GPCR is.
• GPCR Activation Cascade: How GPCRs activate a signaling cascade and how the signaling cascade is terminated.
• Second Messengers:
  • Understanding different types of second messengers and how they are generated.
• Signal Amplification:
  • Understanding signal amplification via GPCRs through examples like adrenaline and cAMP signaling.
• Diversity of GPCR Signaling:
  • Understanding 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 the body responds to sudden short-term stress.
• Alarm Phase:
  • Mobilization of glucose reserves
  • Changes in circulation
  • Increased heart and respiratory rates
  • Increased energy use by all cells
• Adrenaline (Epinephrine) and Noradrenaline:
  • Immediate short-term response to crises and how adrenaline (epinephrine) works.

Adrenaline and GPCRs

• Adrenaline binds to GPCRs.
• Mobilization of glucose reserves in the liver and muscle.
• Increased heart muscle contraction.
• Adrenaline activates tissue-specific GPCRs, leading to the production of cAMP.

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 variety of signaling molecules:
  • Proteins, neurotransmitters, peptide hormones, ions, vitamins, metabolites, photons, fatty acids, 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 (seven-pass transmembrane domain receptors).
• 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).

Heterotrimeric G-Proteins

• Composed of three subunits: α, β, and γ.
  • α and γ subunits are attached to the membrane.
• In the unstimulated (resting) state, the α-subunit has GDP bound, thus is inactive.
• Upon receptor activation, exchange of GDP with GTP activates the α-subunit.
  • The α-subunit dissociates from Gβ and Gγ.
• Hydrolysis of GTP returns everything to the 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.
• Ion Channels: Affects ion flux into cells.
• Tubulin: Affects microtubule dynamics.

Signaling via GPCR and Second Messengers

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

Key Concept: Activated Gα Subunits and Second Messengers

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

Second Messengers

• 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 signaling molecules:
  • Removal or degradation terminates the cellular response.

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 and Free Radicals:
   • e.g., nitric oxide – can signal throughout cell and even to neighboring cells.

• Some second messengers like calcium can also be produced in response to stimuli from within the cell.

cAMP as a Second Messenger

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

Epinephrine-Stimulated cAMP Synthesis

• Epinephrine binding to beta-adrenergic receptor stimulates adenylyl cyclase via G-protein.
• Adenylyl cyclase converts ATP to cAMP.
• Increased heart rate, dilation of skeletal muscle blood vessels, breakdown of glycogen to glucose.

GPCR and Signal Amplification

• Hydrophilic signaling molecules often in low concentration.
• Activate relatively few receptors.
• Signal transduction can generate signal amplification.
• Adrenaline causes the breakdown of glycogen stores.
• One molecule of epinephrine binding to receptor leads to 108 molecules of glycogen being converted to glucose-1-phosphate which is then converted to glucose.

GPCR and Inositol Phospholipid Signaling

• Effector = phospholipase C.
• Cleaves PIP2 to give IP3 and diacylglycerol (DAG), which act as second messengers.
• IP3 acts on Ca^{2+} channels 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 vessels causes the 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 signaling leads to relaxation of smooth muscle cells that surround blood vessels, allowing vessels to dilate.
• Nitric oxide has a 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.

G-Protein Diversity

• G-proteins are characterized by their α subunits, which are further grouped into different families.

Specificity of Signaling

Hormone Activation of GPCRs

• Many water-soluble hormones activate GPCRs.
• Note: Epinephrine can induce distinct signal transduction pathways depending on GPCR and G-protein types!

Conclusions

• GPCR signaling 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 as 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.
• The majority are agonists (activate receptors) targeting metabolism.
• Big interest in the cancer field as emerging anti-cancer drugs.
• GPCRs represent frequent off-targets for drugs.
  • Appetite suppressant drug fenfluramine (Fen-Phen) was popular to combat obesity but was withdrawn by the 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!