G Protein Coupled Receptors

G Protein Coupled Receptors (GPCRs)

Overview

  • GPCRs are a major class of receptors involved in cell signaling.
  • Last week's focus was on enzyme-linked receptors, particularly receptor tyrosine kinases (e.g., EGFR, HER2).
  • Today's focus is on GPCRs, with ion channel receptors to follow this afternoon.

GPCR Activation and Signaling Cascade

  • Ligand binding activates GPCRs, initiating a signaling cascade.
  • Second messengers propagate the signal; these can be diverse molecules (gases, ions) and amplify the signal. Unlike protein kinases, they don't necessarily have to be proteins.
  • Example: Adrenaline (epinephrine) and cAMP signaling.
  • Key concept: GPCR signaling enables strong amplification of the signal from a single ligand-receptor interaction.

Adrenaline and the Fight or Flight Response

  • The fight or flight response is an immediate, short-term reaction to acute stress.
  • Triggers release of adrenaline/noradrenaline (epinephrine) that rapidly reach target cells.
  • Results: Increased heart rate, heightened perception, etc.
  • Mechanism: Adrenaline activates GPCRs on target cells.
  • Stress activates brain signaling, leading from the hypothalamus to the adrenal medulla, which releases adrenaline/noradrenaline.
  • Adrenaline and epinephrine are used interchangeably.
Target Cell Responses:
  • Mobilization of glucose reserves for energy.
  • Changes in circulation to increase oxygen supply (increased heart and respiratory rates).
  • Overall, aims for a sharp burst of energy to cells.

GPCR Mechanism on Target Cells

  • Receptors (e.g., beta-adrenergic receptors in liver/muscle cells) are in the plasma membrane.
  • Ligand (epinephrine) binding activates a G protein (a GTPase).
  • G protein is a complex of alpha, beta, and gamma subunits.
  • Activation involves GDP exchange for GTP on the alpha subunit.
  • GPCR acts as a guanine exchange factor (GEF).
  • Activated GTPase causes a conformational change in an effector enzyme (e.g., adenylyl cyclase in adrenaline signaling).
  • Adenylyl cyclase generates cyclic AMP (cAMP) from ATP – this is a key amplification step.
  • cAMP activates protein kinase A (PKA).
  • PKA phosphorylates downstream proteins, leading to functional effects (e.g., increased glucose reserves via inhibiting glycogen synthesis and promoting glycogen breakdown).
Heart Cell Response:
  • Beta-adrenergic receptor in the plasma membrane.
  • Epinephrine activates the associated GTPase, which activates adenylyl cyclase.
  • cAMP production increases heart muscle contraction, increasing heart rate.
  • Different cell types have slightly different receptor isoforms and GTPase proteins for specific responses.

General Characteristics of GPCRs

  • Largest family of cell receptors: >800 in humans.
  • Many more in animals with a strong sense of smell (smell receptors are often GPCRs).
  • Activated by diverse molecules: neurotransmitters, peptide hormones, proteins, ions, vitamins, photons (in the eye).
  • Involved in numerous physiological processes in almost every cell and tissue.
  • GPCRs are seven-pass transmembrane domain proteins.
  • Ligand binding changes receptor conformation, enabling interaction with and activation of the G protein.

G Protein Activation Cycle

  • G protein (alpha, beta, gamma subunits) is near the GPCR.
  • In the inactive state, the G protein is bound to GDP.
  • Ligand binding to GPCR causes a conformational change, promoting GPCR binding to the G protein.
  • GPCR acts as a GEF, inducing GTP binding instead of GDP.
  • Signal is switched off via GTP hydrolysis back to GDP.
  • Two of the G protein subunits (gamma and alpha) are tethered to the plasma membrane.

Detailed Mechanism of GPCR Activation

  • Resting state: GPCR is inactive, the heterotrimeric G protein ($\alpha$, $\beta$, $\gamma$ subunits) is bound to GDP, and the effector protein (e.g., adenylate cyclase) is inactive.
  • Activation: Ligand binding changes GPCR shape, creating a binding site for the G protein complex.
  • The GPCR acts as a GEF, facilitating GDP to GTP exchange on the $\alpha$ subunit.
  • The activated $\alpha$ subunit-GTP dissociates from the $\beta\gamma$ subunits.
  • The $\alpha$ subunit-GTP then binds to and activates the effector protein.
  • The effector enzyme generates second messenger molecules (e.g., cAMP).
  • GTP hydrolysis deactivates the $\alpha$ subunit, which reassociates with the $\beta\gamma$ subunits, ready for the next signal.

Effector Proteins

  • Adenylyl cyclase is one example of an effector enzyme.
  • Enzymes are the most common effector proteins.
  • Other effector proteins include ion channels.
  • Effector proteins are usually activated by G proteins, but some can be inhibited.

Second Messengers

  • Second messengers are activated/produced after GPCR activation and effector enzyme activation.
  • Examples:
    • cAMP (involved in adrenaline signaling).
    • cGMP.
    • Ions (e.g., calcium).
    • Membrane lipid derivatives (inositol triphosphate/IP3 and diacylglycerol/DAG).
    • Gases (e.g., nitric oxide).
  • Activated G alpha subunits associate with and activate effector enzymes, leading to second messenger production.
  • Protein kinases can also act as second messengers (as seen with receptor tyrosine kinases).
  • Cells maintain a basal level of second messengers, but ligand binding causes a large increase needed to propagate the signal.
  • Second messengers can be quickly produced and degraded, allowing for short-lived signaling.
Cyclic AMP
  • Adenylyl cyclase uses ATP to generate cyclic AMP.
  • Amplification occurs as many cAMP molecules are produced from one activated enzyme.
  • cAMP activates protein kinase A (PKA) by binding to its regulatory subunit, leading to kinase activation.
  • One cAMP activates one PKA, but PKA can then phosphorylate multiple downstream proteins.
  • cAMP can be rapidly degraded.

Epinephrine Receptor Detailed Example

  • Epinephrine binds to the beta-adrenergic receptor.
  • This activates the trimeric G protein, inducing GDP to GTP exchange on the alpha subunit.
  • The alpha subunit-GTP interacts with adenylyl cyclase.
  • Adenylyl cyclase produces cAMP from ATP.
  • This leads to increased heart rate, dilation of skeletal muscle blood vessels, and glycogen breakdown to glucose.

Amplification in GPCR Signaling

  • Receptor to G protein: One receptor can activate multiple G proteins ($\approx$100-fold increase).
  • G protein to effector enzyme: one-to-one.
  • Effector protein to cAMP: One enzyme can generate many cAMP molecules ($\approx$100-fold increase).
  • cAMP to PKA: One-to-one.
  • PKA to downstream substrates: Each kinase can phosphorylate multiple copies of multiple substrates ($\approx$10-fold increase).
Example Cascade
  • Inactive phosphorylase kinase is phosphorylated by activated PKA, becoming active.
  • Active phosphorylase kinase phosphorylates inactive glycogen phosphorylase, activating it.
  • Activated glycogen phosphorylase converts glycogen to glucose-1-phosphate.

Visualizing cAMP Production

  • Cultured neurons stimulated with serotonin show localized G protein coupled receptors.
  • Serotonin binding activates the GTPase and then the effector enzyme, resulting in cAMP production.
  • A dye changes color with cAMP production, showing a burst of cAMP at receptor sites within 20 seconds.
  • The entire activation of the second messenger is very fast, much faster than methods like a 10-minute phosphorylation of MAP kinase. cAMP is upstream in GPCR signaling.

IP3 and DAG as Second Messengers

  • PIP2 (PI4,5-bisphosphate) is a molecule in the plasma membrane.
  • Histamine (in inflammatory responses) binds to its GPCR, activating the G protein.
  • The G alpha subunit-GTP activates phospholipase C (the effector enzyme).
  • Phospholipase C cleaves PIP2 into diacylglycerol (DAG) and inositol triphosphate (IP3).
  • IP3 acts as a second messenger to activate calcium channels on the endoplasmic reticulum (ER).
  • Calcium is released into the cytoplasm and acts as a second messenger, binding to protein kinase C (PKC).
  • PKC also needs DAG for activation, resulting in phosphorylation of downstream substrates.
  • This process involves multiple second messengers: IP3, calcium, and DAG.

Nitric Oxide as a Second Messenger

  • Acetylcholine binds to its receptor on endothelial cells.
  • This activates a trimeric G protein, leading to phospholipase activation.
  • Phospholipase cleaves PIP3 into IP3.
  • IP3 causes calcium release from the ER via activation of ion channels.
  • Calcium activates nitric oxide synthase, which produces nitric oxide from arginine.
  • Nitric oxide rapidly diffuses through the plasma membrane into smooth muscle cells (no receptors needed).
  • In smooth muscle cells, nitric oxide activates guanylyl cyclase, converting GTP to cyclic GMP (cGMP).
  • cGMP acts as a second messenger, causing smooth muscle relaxation and vasodilation.
  • Involves multiple second messengers: IP3, calcium, nitric oxide, and cGMP.