Lecture 6: GPCRs II

Aims

To describe the signalling that occurs through GPCRs, the role of second messengers in signal amplification and coordinating cellular responses and mechanisms used to limit signalling through these receptors.

Learning Objectives

●      List the key second messenger systems regulated by GPCRs and describe the regulation of their synthesis and breakdown

●      Give examples of effector proteins regulated by GPCRs

●      Describe desensitisation of GPCRs and its implications for medicine

Overview of G-Protein Coupled Receptor (GPCR) Signaling and Second Messengers

• Second messengers are small molecules responsible for carrying signals inside cells.

• Activated G-proteins regulate the activities of enzymes that control the levels of these second messengers.

• Second messengers are categorized into two primary types based on their solubility:

  • Hydrophobic lipids: These molecules are confined to the membrane where they are generated (e.g., Diacylglycerol).

  • Small soluble molecules: These molecules diffuse through the cytoplasm (e.g., $cAMP$, $cGMP$, $IP_3$, and $Ca^{2+}$).

• The specific message conveyed is encoded in the concentration and the frequency of changes in that concentration, which are determined by:

  • The rate of production.

  • The rate of diffusion.

  • The rate of removal.

Functional Classification of Trimeric G-Proteins and Effectors

• Trimeric G-protein effectors include enzymes that create second messengers and ion channels whose gating is regulated directly by $\beta\gamma$ subunits or indirectly by second messengers.

• $G_{i\alpha}$ Family (7 human members):

  • Receptors: $\alpha$-Adrenergic amines, acetylcholine, chemokines, and various neurotransmitters.

  • Effectors: Inhibits adenylyl cyclase, opens potassium ($K^+$) channels, and closes calcium ($Ca^{2+}$) channels.

• $G_{s\alpha}$ Family (3 human members):

  • Receptors: $\beta$-Adrenergic amines, hormones (corticotropin, glucagon, parathyroid, thyrotropin, etc.).

  • Effectors: Stimulates adenylyl cyclase to produce $cAMP$; involves receptor kinase.

• $G_{q\alpha}$ Family (5 human members):

  • Receptors: $\alpha$-Adrenergic amines, acetylcholine, and various neurotransmitters.

  • Effectors: Activates phospholipase $C\beta$ ($PLC\beta$) to produce $IP_3$, which triggers the release of $Ca^{2+}$.

• $G_{12/13\alpha}$ Family (2 human members):

  • Receptors: Thrombin and others.

  • Effectors: Activates Rho and other signaling proteins.

• $G_{t\alpha}$ (Transducin, 2 human members):

  • Receptors: Rhodopsin (absorbs light).

  • Effectors: Activates $cGMP$ phosphodiesterase to break down $cGMP$; involves receptor kinase.

• $G_{olf\alpha}$ Family:

  • Receptors: Odorant receptors.

  • Effectors: Activates adenylyl cyclase; involves receptor kinase.

The Cyclic AMP (cAMP) Second Messenger System

• Adenylyl Cyclase (Adenylate Cyclase):

  • There are 10 known isoforms of this membrane-anchored enzyme.

  • It is activated by $G_{s\alpha}$ and inhibited by $G_{i\alpha}$.

• Signaling Steps:

  1. A ligand binds to the GPCR, activating the G-protein.

  2. The $\alpha$ subunit moves and binds to adenylyl cyclase in the membrane.

  3. The activated enzyme catalyzes the formation of $cAMP$ from $ATP$.

  4. $cAMP$ (the second messenger) activates Protein Kinase A (PKA).

  5. PKA phosphorylates and activates target proteins.

  6. This initiates a specific cellular response.

• Example: Adrenaline (Epinephrine) and Glycogen Breakdown:

  • Adrenaline binds to a $\beta_2$ adrenoceptor (a GPCR) in the liver or skeletal muscle.

  • The GPCR undergoes a conformational change, causing the $G_{\alpha}$ subunit to swap $GDP$ for $GTP$.

  • Adenylyl cyclase is activated, converting $ATP$ to $cAMP$.

  • $cAMP$ binds to inhibitor proteins on PKA, releasing the active PKA subunits.

  • A phosphorylation cascade occurs: PKA phosphorylates Phosphorylase Kinase, which then activates Glycogen Phosphorylase.

  • This leads to the breakdown of glycogen into glucose for energy.

• Mechanisms for Terminating cAMP Signaling:

  1. Agonist (ligand) dissociates from the receptor.

  2. The intrinsic $GTPase$ activity of $G_{s\alpha}$ hydrolyzes $GTP$ back to $GDP$.

  3. $cAMP$ is broken down into $AMP$ by the enzyme phosphodiesterase.

  4. Phosphatases dephosphorylate the activated enzymes.

The Cyclic GMP (cGMP) Second Messenger System

• The $cGMP$ system is similar to the $cAMP$ system but utilizes guanylate cyclase.

• Guanylate cyclase can be either membrane-bound (receptor-bound) or "free" in the cytoplasm.

• The enzyme converts guanosine triphosphate ($GTP$) to $3', 5'-cyclic\,guanosine\,monophosphate$ ($cGMP$).

• Functions of cGMP Signaling:

  • Phototransduction (vision).

  • Smooth muscle relaxation.

  • Platelet inhibition.

  • Gene transcription regulation.

  • $Ca^{2+ }$ release.

• $cGMP$ levels are regulated by the rate of production via guanylate cyclase and removal via phosphodiesterase.

Phospholipase C (PLC) and Membrane Lipid Signaling

• GPCRs can activate $PLC\beta$ to target membrane lipids and generate two types of second messengers from the precursor Phosphatidylinositol $(4, 5)$-bisphosphate ($PIP_2$):

  • Inositol trisphosphate ($IP_3$): A water-soluble molecule that diffuses through the cytoplasm to targets such as the endoplasmic reticulum (ER).

  • Diacylglycerol (DAG): A hydrophobic molecule that remains within the plasma membrane.

• Lipid Kinases: These enzymes add phosphate groups to lipids. For example, they can add a phosphate to DAG to create phosphatidic acid, or to phosphatidylinositol (PI) to generate $PIP$, $PIP_2$, or $PIP_3$.

• Protein Kinase C (PKC):

  • PKCs are serine/threonine (Ser/Thr) kinases.

  • They are activated by DAG (via the $C1$ domain) and $Ca^{2+}$ (via the $C2$ domain).

  • PKC can provide both positive and negative feedback.

  • Phosphorylation of $PLC\beta$ by PKC provides negative feedback, making the signal transient.

  • DAG binding induces the dissociation of an intramolecular pseudosubstrate, activating the kinase.

Calcium (Ca2+) as a Second Messenger

• Cytosolic Concentration: In resting cells, cytosolic $[Ca^{2+}]$ is maintained at a very low level, approximately $100\,nM$, by $ATP$-driven $Ca^{2+}$ pumps.

• Mechanisms of Calcium Influx:

  • IP3-mediated release: $IP_3$ binds to ligand-gated $Ca^{2+}$ channels on the ER membrane, causing an influx of $Ca^{2+}$ into the cytoplasm from internal stores.

  • Store-Operated Calcium Entry (SOCE): When ER calcium stores are depleted, $STIM$ dimers (acting as calcium sensors) move and bind to $ORAI$ channels in the plasma membrane. This allows $Ca^{2+}$ to move from the extracellular space into the ER lumen to refill the stores.

• Applications: Ca2+ imaging using fluorescent tags and genetically engineered animal models allows for the in vivo recording of calcium signaling to study disease mechanisms and develop new drugs.

Desensitization of GPCRs

• Desensitization is a critical regulatory mechanism to prevent overstimulation of a pathway.

• Key Regulators:

  • GPCR Kinases (GRKs): Phosphorylate the receptor.

  • $\beta$-arrestin: Binds to the phosphorylated receptor.

• Functions of Desensitization:

  1. Physically prevents the G-protein from binding to the receptor.

  2. Promotes receptor internalization, where the receptor is either degraded or recycled back to the membrane.

• Clinical and Pathological Implications:

  • Positive Aspect: Prevents uncontrolled growth (e.g., in cancer).

  • Negative Aspect: Leads to Tachyphylaxis, which is a rapidly diminishing response to successive doses of a drug, rendering the treatment less effective.

  • GPCR signaling pathways are vital targets for medicine, and understanding their molecular mechanisms is essential for identifying mutations and developing therapies.