L5 - GPCR Pt. 2

Signal Transduction: Signaling of Inositol Phospholipids

Overview of Phospholipids

• Phospholipids: Composed of a glycerol backbone with ester bonds.

• Can be hydrolyzed by phospholipases, leading to the release of intracellular second messengers.

• Examples of receptors that utilize this pathway:

  • α1-adrenergic receptor

  • Muscarinic M1 receptor

  • Serotonin 5-HT2 receptor

  • Angiotensin AT1 receptor

• Note: Do not memorize the specific phospholipase cleavage sites.

Phosphatidylinositol Pathway

• Involves the Gαq subunit.

• General Scheme:

  • Extracellular Binding: Signal binds to G-protein-coupled receptor (GPCR).

  • Activation of Phospholipase C (PLC): Hydrolyzes membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers:

  • Inositol 1,4,5-triphosphate (IP3)

  • Diacylglycerol (DAG)

  • Diagram illustrating the conversion:

    • $ext{PLC} <br>ightarrow ext{DAG} + ext{IP3}$

Phospholipase C Hydrolysis

• Breakdown of PIP2 leads to products:

  • Inositol 1,4,5-trisphosphate (IP3):

  • Characteristics:

    • Water soluble.

    • Binds to a receptor on the membrane of the smooth endoplasmic reticulum and mitochondria, resulting in the opening of calcium channels.

  • Diacylglycerol (DAG):

  • Characteristics:

    • Remains membrane-bound.

    • Activates Protein Kinase C (PKC), which then phosphorylates other proteins, altering their activity.

  • Note: This is a distinct isoform of protein kinase compared to that in the cAMP pathway.

Phospholipase C Activation Mechanisms

• Two main activation mechanisms for phospholipase C:

  • G-proteins: The alpha subunit of Gq/11 activates phospholipase C through interaction at its C-terminus.

  • Tyrosine kinase: Domains of phospholipase C interact with tyrosine kinase, becoming phosphorylated, which activates PLC.

Signal Transduction Cascade in the Phosphatidylinositol Pathway

• Intracellular Components:

  • Endoplasmic reticulum

  • IP3

  • Activated Gq alpha subunit

  • PIP2

  • Activated PLC

  • DAG

  • Activated Protein Kinase C (PKC)

  • Calcium ions (Ca2+)

  • G-protein linked receptor

  • Flow of signal: Extracellular to Intracellular.

Protein Kinase C (PKC)

• Inactive Form: Synthesized as a non-phosphorylated precursor.

• Active Form:

  • Associated with the plasma membrane via interaction with DAG.

• Regulation:

  • Partially activated by phosphorylation from phosphoinositide-dependent kinase-1 (PDK1).

  • Fully activated through autophosphorylation.

  • Also regulated by calcium ions (Ca2+).

Protein Kinase C Signaling Pathways

Key Examples of PKC Pathways

• Examples of pathways where PKC plays a critical role:

  • Glucose/glycogen homeostasis

  • Bronchoconstriction

  • Constriction processes

  • Ejaculation processes

  • Secretion mechanisms

Attenuation Mechanisms for PKC

• Multiple methods exist for deactivating PKC:

  • Phosphorylation of DAG

  • Dephosphorylation of PKC

  • Degradation of PKC protein

Signal Transduction Involving G Proteins

Ligands and Function

• Ligands:

  • Epinephrine

  • Norepinephrine

• Locations: Found in adrenal medulla and CNS (synaptic vesicles) and throughout the body.

• Physiological Response: Triggers fight-or-flight response leading to effects like:

  • Sweating

  • Piloerection (hair standing up)

  • Pupil dilation

  • Bronchi dilation

  • Increased heart rate and force of contraction.

Adrenergic Receptors and Second Messenger Changes

Signal Mechanisms of α- and β-Adrenergic Receptors

α-Receptor Signaling

• α1 Activation: Leads to PLC activation through interaction with Gαq.

• α2 Activation: Inhibits adenylyl cyclase and increases PLC activity.

• RGS (Regulator of G-Protein Signaling): Shortens signal duration by increasing GTP hydrolysis associated with α receptors.

β-Receptor Signaling

• Both β1 and β2 adrenergic receptors can activate adenylyl cyclase.

• β2 receptors: Have a component of Gαi, which can modulate signaling.

• β-arrestin and RGS: Play roles in attenuating and decreasing β-receptor signaling.

Epinephrine Concentration-Dependent Effects

• Low Concentrations: Activate predominantly β1 and β2 receptors.

• High Concentrations: Effects of α1 receptors become dominant.

  • β1 receptors:

  • Positive inotropy (↑ cardiac contractility)

  • Positive chronotropy (↑ heart rate)

• Clinical Applications:

  • Used therapeutically for conditions such as anaphylaxis and acute asthma exacerbations.

  • β2 receptor Activation Effects:

  • Vasodilation leading to reduced peripheral vascular resistance and diastolic blood pressure.

  • Increased blood flow to skeletal muscles.

  • Bronchial smooth muscle relaxation.

  • Enhanced metabolic activity with catabolic effects.

Norepinephrine Effects Overview

• Agonistic Effects: Targets α1, α2, and β1 adrenergic receptors, with no effect on β2 receptors.

• Physiological Effects:

  • Increases both systolic and diastolic pressure and total peripheral resistance.

  • Chronotropic effects tempered by vagal response.

  • Augments stroke volume without changing overall cardiac output.

Dopamine Receptor Types and Effects

Dopamine and Pharmacology

• Dopamine: The precursor of norepinephrine.

• Administration notes: Poor entry into CNS systemically.

• Concentration-Dependent Effects:

  • Low Dose Effects:

  • Primarily vasodilation via D1 receptors in renal, mesenteric, and coronary vasculature.

  • High Dose Effects:

  • Acts as a positive inotrope in the heart with β1 adrenergic receptor activity.

  • At even higher doses, shows α1 adrenergic agonist activity leading to vasoconstriction.

Acetylcholine Receptors

Muscarinic ACh Receptor Activities

Regulation of GPCR Signaling

• GTPase Activity: Regulator of G-Protein Signaling (RGS) increases GTP hydrolysis into GDP.

• Desensitization: Involves phosphorylation of the receptor; allows binding of beta-arrestin, preventing further interaction with trimeric G-protein.

• Receptor Degradation/Recycling:

  • Internalization processes lead to either fusion with lysosomes for degradation or recycling back to the plasma membrane.

Desensitization Mechanism Overview

• G-Protein Receptor Kinase (GRK): Aids in receptor desensitization.

  • Targets the receptor for endocytosis, leading to a reduction in cell surface receptor numbers.

  • Phosphorylates GPCR at specific amino acids, which increases beta-arrestin binding.

  • Beta-arrestin binding inhibits signaling pathways by preventing association with G-proteins.

Beta-Adrenergic Receptors and Competing Second Messenger Systems

• Agonism of β1AR: Leads to Gαs activation and involvement of GRK.

• Chronic Stimulation: Results in excess GPCR signaling; GRK serves a cardioprotective role by downregulating signaling responses to agonists.

• Transcriptional Control: Manages the transactivation of pathways related to receptor recycling and signaling.

Role of β-Arrestins in Desensitization

• β-arrestin Function: Promotes GPCR incorporation into clathrin-coated pits in the membrane, facilitating internalization.

• Dynamin: Protein responsible for mediating the internalization of GPCR and the associated plasma membrane segment.

• Recycling and Regulation: Release of β-arrestin promotes the recycling of receptors back to the cell surface for subsequent signaling use.