HLSC 2P09 Signal Transduction: Receptors & Overview

• Signal transduction, for HLSC 2P09, updated Feb 2024

Signal Transduction Overview

• Signal transduction is the process where information from the extracellular environment gets conveyed to the cell's interior.
• Signaling molecules like hormones and growth factors, carry information to specific effector molecules within the cell.
• Ligands, or signaling molecules, combine with ligand receptors to initiate a cascade of reactions.
• Only cells expressing the receptor for a specific ligand will respond to that ligand.

Receptor Types

• Lipophilic signal molecules diffuse through the cell membrane and bind to cytosolic or nuclear receptors, leading to slower responses related to gene activity changes.
• Lipophobic signal molecules bind to receptors on the cell surface, triggering rapid cellular responses.
• There are various receptor types: 7 Transmembrane (TM) receptors that interact with GTP-binding (G) proteins; single transmembrane receptors with intrinsic kinase activity (tyrosine, serine threonine); single transmembrane receptors that interact with tyrosine kinase; ion channels; cytosolic-nuclear receptors.
• Examples of 7 TM receptors include Glucagon and Vasopressin (ADH).
• Single TM Receptors include insulin and growth factors.
• Intracellular receptors include: Glucocorticoids, Mineralcorticoids, Steroids (sex hormones) and Thyroid hormone.

Second Messenger Hypothesis

• The Second Messenger Hypothesis describes the signalling cascade where a signalling molecule (hormone) binds to a receptor in the plasma membrane, triggering a cascade of events which activate a secondary messenger to illicit a response.

Second Messengers

• Common second messengers include: 3'-5' AMP (cAMP), Diacyglycerol (DAG), Ca++, and Inositol 1,4,5 trisphosphate (IP3).

Second messenger pathways

• Calcium ions bind to calmodulin altering enzyme activity or binding to other proteins causing exocytosis, muscle contraction and cytoskeleton movement.
• cAMP activates protein kinases especially protein kinase A or binds to ion channels, altering channel opening.
• cGMP activates protein kinases especially protein kinase G or binds to ion channels, altering channel opening.
• IP3 Releases Ca2+ from intracellular stores.
• DAG activates protein kinase C (PK-C) to phosphorylate proteins.

Protein Phosphorylation

• Protein phosphorylation is a modification of many cellular proteins and it is an important step in signal transduction pathways.
• The net phosphorylation levels are determined by the activity of kinases and phosphatases.

Kinases and Phosphatases

• Kinases add phosphate groups to serine/threonine or tyrosine residues while Phosphatases remove them.
• Kinases include Serine/Threonine kinases and Tyrosine kinases.
• Phosphatases include Serine/Threonine Phosphatases (PSPs) and Tyrosine Phosphatases (PTPs).

G-Protein Coupled Receptors

• 7 TM receptors, also known as "serpentine" receptors, interact with GTP-binding (G) proteins and span the plasma membrane 7 times.
• These receptors are associated with heterotrimeric G-Proteins, which have three subunits: alpha, beta, and gamma.
• The ligand (hormone or signaling molecule) binds to the 7 TM Receptor causing GTP to replace GDP on the alpha-subunit binding site, making it dissociate from the beta and gamma subunits.
• The activated alpha subunit then activates an effector molecule like Adenyl Cyclase or Phospholipase C.

Families of G proteins

• Gs stimulates Adenylyl cyclase (AC)
• Gi inhibits AC
• Gq stimulates Phospholipase C (PLC)
• G12 regulates the sodium/hydrogen exchanger (NHE)

Toxin Interference

• Cholera toxin causes ADP-ribosylation of Gα(arg), abolishing GTPase activity.
• This locks Gsα in an active monomeric state leading to severe watery diarrhea.
• Pertussis toxin causes ADP-ribosylation of Giα(cys), inhibits GDP/GTP exchange and locks Gia in an inactive heterotrimeric state.

G-Protein and Adenylyl Cyclase

• The ligand binds to a 7TM receptor, this activates a G protein and causes GTP to replace GDP on the binding site of the alpha subunit.
• The activated alpha subunit then activates an effector molecule called Adenylyl Cyclase.
• Adenylyl cyclase converts ATP to cyclic AMP (cAMP).
• cAMP then activates Protein Kinase A (PKA).
• Protein kinase A phosphorylates other proteins, leading to a cellular response

Regulation of cAMP levels

• cAMP is converted to AMP by phosphodiesterase.

G-Protein and Phospholipase C

• The ligand binds to a 7TM receptor, this activates a G protein and causes GTP to replace GDP on the binding site of the alpha subunit.
• The activated alpha subunit then activates an effector molecule called Phospholipase C (PLC).
• PLC converts phosphatidylinositol 4,5 P2 (PIP2) into IP3 + DAG.
• IP3 releases calcium into the cytosol from the endoplasmic reticulum.
• DAG activates protein kinase C.
• Increased intracellular Ca++ activates CaM kinase.
• Kinase activation and protein phosphorylation leads to a response.

Ion Channels

• Signaling molecules can regulate ion channels in the plasma membrane.
• Receptor-channels open or close in response to signal molecule binding.
• Some channels are directly linked to G proteins.
• Other ligand-gated channels respond to intracellular second messengers.

Calcium as a Second Messenger

• Intracellular calcium levels are maintained at 0.01-0.1 uM (10-7 M) at resting conditions.
• Extracellular calcium levels are much higher at approx. 2-5mM (10-3 M).
• Calcium channels in PM, voltage gated Ca++ channels in PM and IP3 gated Ca++ channels in ER, and ryanodine receptors in SR cause release into the cytosol.
• Ca++ ATPase in PM and in ER / SR causes sequestration out of the cytosol.
• Increased intracellular Ca++ can bind to proteins (Calmodulin, Troponin C) or remain free to activate PKC or cause exocytosis.

Tyrosine Kinase Receptors

• Tyrosine kinase receptors are membrane receptors that have an intrinsic tyrosine kinase activity in their intracellular loop.
• General receptors activate the PI3K-Akt and Ras-MAPK signaling pathways.

PI3K-Akt cascade signaling pathway

• The PI3K-Akt signaling cascade operates in all cells and is important for cell proliferation, survival and inhibition of apoptosis.
• Ligand binding causes a conformational change in the receptor and the receptor is auto-phosphorylated, which increases tyrosine kinase activity.
• The activated receptor attracts phosphatidylinositol 3-kinase (PI3K) that converts membrane phospholipids to 3-phosphoinositides (PIP3).
• PIP3 binds and activates phosphoinositide-dependent kinase (PDK), which then phosphorylates and activates Akt.
• Activated Akt then phosphorylates intracellular proteins to produce cellular responses such as increased cell proliferation and survival.

PI3K-Akt cascade in Insulin Binding

• With insulin, the intracellular substrate is insulin receptor substrate-1 (IRS-1).
• IRS-1 binds to the receptor and is phosphorylated by its kinase activity.
• Phosphorylated IRS-1 attracts PI3K and activates it.
• PI3K converts membrane phospholipids to PIP3 which activates PDK.
• PDK then phosphorylates and activates Akt, leading to metabolic responses like GLUT4 glucose transporter translocation.

Ras-MAPK Signaling Cascade

• The ligand (growth factor) binds to receptor and increases the its tyrosine kinase activity within the intracellular receptor.
• Grb2-SOS exists as a complex.
• Grb2 via its SH2 domains binds to phosphorylated/activated receptor.
• SOS is a GTP/GDP exchanger, SOS brings Ras into a GTP-bound and active form
• Activated Ras binds to Raf kinase and activates it.
• Raf kinase phosphorylates MAP kinase kinase (MEK) and activates it.
• Activated MAP kinase kinase (MEK) phosphorylates MAP kinase (MAPK) and activates it.
• MAP kinase (MAPK) phosphorylates proteins which leads to cellular responses.

Ras-MAPK Signaling Cascade in Insulin Binding

• IRS-1 binds to the receptor and is phosphorylated by its kinase activity.
• Phosphorylated IRS-1 attracts the Grb2-SOS complex.
• SOS brings Ras into a GTP-bound form and activates it.
• Activated Ras binds to Raf kinase and activates it.
• Raf kinase phosphorylates MAP kinase kinase (MEK) and activates it.
• Activated MAP kinase kinase (MEK) phosphorylates MAP kinase (MAPK) and activates it.
• MAP kinase (MAPK) which phosphorylates proteins and leads to a range of responses.

Receptors associated with TKs and the JAK-STAT Pathway

• Growth Hormone binds to its receptor
• This causes a conformational change that brings the two JAKS associated with the receptor together; JAK is a tyrosine kinase.
• JAK autophosphorylation and activation follow.
• JAK phosphorylates tyrosine residues of the receptor.
• STAT (signal transducer and activator of transcription) binds to the phosphorylated tyrosine residues and is phosphorylated as well.
• Phosphorylated STAT dissociates, dimerizes, and translocates to the nucleus to regulate gene transcription.

Steroid Hormone Mechanism

• Hormone enters the cell and binds with a specific receptor.
• Hormone/receptor diffuses into the nucleus to activate gene transcription forming messenger RNA (mRNA).
• The mRNA diffuses into the cytoplasm and promotes translation to form new proteins.
• Steroid-hormones bind to intracellular receptors activing Hormone Response Elements on DNA to modify gene transcription.
• GRE (glucocorticoid), ERE (estrogen), and TRE (thyroid hormone) are Hormone Response Elements.

Hormone Action

• Signaling molecules or hormones act by changing: cell membrane permeability, levels of a metabolite, enzyme function, gene transcription, protein-protein interaction, cellular localization of substrate, or cytoskeletal arrangement.

Amplification

• Amplification during signal transduction increases the effect of the signaling molecule.

Crosstalk

• Crosstalk in signal transduction provides multiple pathways and mechanisms of action.

Receptor agonists and antagonists

• Receptor agonists activate receptors eliciting a response.
• Receptor antagonists block receptors and block a response.

Multiple Receptors

• There can be multiple receptors for the same ligand.
• Epinephrine, for example, can bind to different isoforms of the adrenergic receptor: alpha and beta.

Cyclic GMP (cGMP)

• Cyclic nucleotides have been extensively studied as second messengers of intracellular events initiated by activation of many types of hormone and neurotransmitter receptors.
• Peptide hormones activate receptors associated with membrane-bound guanylate cyclase (GC), leading to conversion of GTP to cGMP.
• Nitric oxide (NO) also stimulates cGMP production by activating soluble GC.
• cGMP mediates most of its intracellular effects through specific cGMP dependent protein kinases (PKG).
• Phosphodiesterases (PDE-I-VI) catalyze the degradation of cGMP to guanosine-5'-monophosphate (5'-GMP).
• Stimulation and Inhibition of PDE varies depending on the type.

Cyclic GMP pathway in vasodilation

• Vasoactive drugs elevate calcium in endothelial cells, which activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO).
• Nitric oxide reaches neighboring muscle cells and stimulates guanylyl cyclase to synthesize cyclic GMP, activating protein kinase G and leading to vasodilation.

Explore signal transduction, the process of transferring information from the extracellular environment to a cell's interior, crucial for HLSC 2P09. Learn about signaling molecules, receptor types (lipophilic and lipophobic), and understand rapid vs. slow cellular responses. Discover how ligands combine with receptors to trigger reaction cascades.