GPCRs and Their Signaling Pathways

G-Protein Coupled Receptors (GPCRs)

• Module Objectives:

  • Describe how ligand binding to a GPCR leads to activation of a trimeric G-protein.

  • Understand the two main pathways activated by GPCRs:

    • Phospholipase C (PLC) and Protein Kinase C (PKC)

    • cAMP and Protein Kinase A (PKA)

  • Compare different classes of G-proteins:

    • Monomeric vs. trimeric

    • Inhibitory vs. stimulatory

    • PLC and cAMP pathways

  • Describe how cell function can be altered by stimulating or inhibiting specific GPCRs.

GPCR Activation

• Mechanism:

  • Ligand (signal molecule) binds to the GPCR.

  • This binding activates the G-protein.

  • The activated G-protein then activates an enzyme, triggering a downstream signaling cascade.

Receptor and G-Protein Structure

• GPCR Structure:

  • Characterized by 7 transmembrane domains.

  • Has one site for G-protein binding and one site for ligand binding.

  • There are approximately 100 different GPCRs that bind various ligands like adrenalin and histamine.

• G-Protein Association:

  • The G-protein is located on the cytosolic side of the plasma membrane.

  • It interacts with the GPCR upon activation.

  • G-proteins can be either active or inactive.

G-Proteins

• Types of G-proteins:

  • Trimeric G-proteins: Associated with GPCRs

  • Monomeric G-proteins: Example is RAS G-protein

• Activation:

  • GPCR activates the G-protein upon ligand binding.

  • Active G-proteins proceed to activate other enzymes, initiating downstream signaling.\n

GPCR Signaling Pathways

• Pathway 1: Phospholipase C (PLC) to Protein Kinase C (PKC)

  • Ligand binding to GPCR activates PLC.

  • PLC cleaves phosphatidylinositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3).

  • DAG and IP3 act as second messengers.

• Pathway 2: Adenylate Cyclase to Protein Kinase A (PKA)

  • Ligand binding to GPCR activates Adenylate Cyclase.

  • Adenylate Cyclase converts ATP to cyclic AMP (cAMP).

  • cAMP binds to PKA, activating it.

  • P-PKA goes to the nucleus and activates CREB transcription factor.

  • CREB (cAMP Response Element Binding protein) initiates gene expression by binding to cAMP-responsive genes, altering cell activity.

Adenylate Cyclase Pathway: A Detailed Look

• Steps:

  • Adenylate cyclase is activated by ligand binding to a GPCR.

  • Activated adenylate cyclase converts ATP to cAMP.

  • cAMP binds to PKA and activates it.

  • Activated P-PKA translocates to the nucleus and activates CREB.

  • CREB activates the expression of cAMP-responsive genes.

• Outcome:

  • Altered cell activity via gene expression.

  • The specific genes expressed depend on the signal molecule, the GPCR involved, and the cell type.

cAMP-Mediated Cell Responses

• Examples:

  • Thyroid gland: Thyroid-stimulating hormone (TSH) induces thyroid hormone synthesis and secretion.

  • Adrenal cortex: Adrenocorticotrophic hormone (ACTH) induces cortisol secretion.

  • Ovary: Luteinizing hormone (LH) induces progesterone secretion.

  • Muscle: Adrenaline induces glycogen breakdown.

  • Bone: Parathormone induces bone resorption.

  • Heart: Adrenaline increases heart rate and contraction force.

  • Liver: Glucagon induces glycogen breakdown.

  • Kidney: Vasopressin induces water resorption.

  • Fat: Adrenaline, ACTH, glucagon, and TSH induce triglyceride breakdown.

• Adrenaline (Epinephrine) example:

  • Adrenaline uses the cAMP pathway via GPCRs but has different effects on different tissues.

  • Adrenaline binds to β-adrenergic receptors (GPCR).

  • This leads to increased cAMP and PKA activity, resulting in glucose release from glycogen and inhibiting glycogen synthesis.

Adrenergic Receptors and Adrenaline

• Adrenaline binds to many different adrenergic receptors, all of which are GPCRs.

• Common adrenergic receptor types include α1, α2, β1, and β2.

• Adrenaline production can cause various effects depending on the receptor type and tissue.

GPCRs: Activation and Inhibition

• GPCRs can either activate or inhibit signaling pathways.

• Types of G-proteins:

  • Gi/Go: i = inhibitory, o = other

  • Gs: s = stimulatory

  • Gα: activates adenylate cyclase

  • Gq: activates PLC pathway

• Functional Implications:

  • Gai protein inhibits signaling.

  • Gas protein stimulates signaling.

Targeting GPCRs with Drugs

• Targeting specific receptors allows alteration of a subset of actions.

• Beta-blockers:

  • Antagonize β2 adrenergic receptors.

  • Used in hypertension treatment (e.g., Butoxamine, Propranolol).

• Beta-agonists:

  • Activate β2 adrenergic receptors, like adrenaline.

  • Used in asthma treatment because they cause relaxation in smooth muscle cells (especially bronchial) (e.g., Isoprenaline, Levalbuterol, Metaproterenol).

Questions about G-protein activation

• Which is the active version of this G-protein?

• What is the enzyme called that will inactivate it?

• What is the enzyme called that will re-activate it?

Questions about Growth Factors and G-protein

• If this G-protein is activated by a growth factor a) b) What kind of receptor will it interact with?

• What will be the main result in the cell?

Caffeine and Adenosine Receptors

• Caffeine works by binding to adenosine receptors.

• Adenosine has 4 receptors – A1 and A2A have sedative effects on brain blood vessels dilate, neurons ‘unwind’ Gai or Gas ?

*Mechanism of Caffeine:
*Caffeine does NOT lead to G protein activation.
*Adenosine can’t bind if caffeine present.
*Blood vessels can’t dilate.
*Neurons can’t “wind down”.
*You can’t feel sleepy.

*Napping: Adenosine receptors clear their ligand faster
*20 mins = time for caffeine to get into brain
* = time for adenosine receptors to turn over
*More receptors ready to receive caffeine and not adenosine

Endocannabinoid System and GPCRs

• Receptors:

  • CB1: Mostly neurons, other brain cells at high level; other tissues at low level.

  • CB2: Mostly immune cells at high level; also regulates pain perception.

• Functions:

  • Memory and learning

  • Brain plasticity

  • Neuronal development

  • Thermogenesis

  • Addiction

  • Nociception

  • Energy balance

  • Appetite regulation

  • Digestion

  • Metabolism

  • Motility

  • Fertility

• CB1

  • interacts with Gai/o proteins => what would the main effect be?
    *Mostly neurons, other brain cells at high level; other tissues at low level

*Which receptor would you pick as a target to treat autoimmune disease, without psychoactive effects?

• Signaling complexity in CB1R:

  • G-alpha protein blocks AC and indirectly up-regulates MAPKKK.

  • Beta/gamma proteins activate PI3K/AKT.

  • Ligand binding can activate another pathway = b-arrestin pathway.

  • Some ligands will preferentially activate Ga, and some will activate b-arrestin = “biased”.

• CB2R signaling also up-regulates MAP KKK and the PI3K AKT pathway

• Ligands:

  • Endocannabinoids (naturally synthesized by us):

    • Anandamide (AEA)

    • 2-arachidonoyl-glycerol (2-AG)

  • Phytocannabinoids (naturally synthesized by plants):

    • THC = CB1 > CB2

    • CBD = antagonize CB1/CB2, bind to other receptors

    • and another 120+ compounds 

*Targeting CB R signalling with medicinal cannabis
*delta-9-tetrahydrocannabinol 27 mg/ml (from Tetranabinex®

• Cannabis sativa L. extract) and cannabidiol 25 mg/ml
  (from Nabidiolex® - Cannabis sativa L. extract)

• Buccal Spray
  *Adjunctive treatment for the symptomatic reef of
  neuropathic pain in multiple sclerosis in adults