Unit 3 lecture 2

Overview of Cell Signaling and Receptor Classifications

• Cellular communication relies on three core stages:

  • Reception: An extracellular signaling molecule (ligand) specifically binds to its complementary receptor located on the plasma membrane or inside the cell.

  • Transduction: A sequence of intracellular biochemical events and molecular interactions that relays and amplifies the external signal into the cell interior.

  • Response: The ultimate downstream cellular alteration resulting from the signaling pathway, such as an alteration in gene transcription, entry into mitosis, or metabolic shifts.

• Receptors fall into distinct structural and functional classes:

  • Ligand-Gated Ion Channels: Membrane-spanning protein complexes forming an aqueous pore that opens or closes upon ligand binding, allowing the facilitated diffusion of specific ions without requiring intrinsic enzymatic or catalytic activity.

  • G-Protein Coupled Receptors (GPCRs): Integral membrane proteins characterized by exactly seven transmembrane $\alpha$-helices coupled intracellularly to a heterotrimeric G protein.

  • Receptor Tyrosine Kinases (RTKs): Cell surface receptors possessing intrinsic enzymatic (kinase) activity; upon ligand binding, RTK monomers dimerize and trans-autophosphorylate specific intracellular tyrosine residues.

  • Intracellular Receptors: Non-membrane-bound receptors situated in the cytoplasm or nucleus that bind hydrophobic ligands (such as steroids) capable of diffusing freely across the lipid bilayer.

Ligand-Gated Ion Channels and Membrane Potentials

• Structural requirements of ion channels:

  • A single transmembrane helix cannot form an ion channel because it cannot create an aqueous passageway across the hydrophobic core of the lipid bilayer.

  • Channel formation requires multiple transmembrane segments or multi-subunit assemblies that come together to enclose a central hydrophilic pore.

  • In the absence of a ligand, the channel remains in a closed conformation; ligand binding induces a conformational change that opens the pore.

• Membrane potential alterations:

  • The baseline resting membrane potential of a living cell is negative on the inside relative to the outside.

  • Depolarization: The influx of cations (positively charged ions) into the cytosol reduces the electrical gradient, making the interior of the cell more positive.

  • Hyperpolarization: The influx of anions (negatively charged ions) increases the electrical gradient, making the cell interior more negative.

  • Ion movement across these channels occurs purely via facilitated diffusion down the electrochemical gradient, entirely devoid of direct catalytic or enzymatic activity.

Receptor Tyrosine Kinases (RTKs) and the RAS-MAPK Cascade

• RTK activation mechanism:

  • Ligand binding (such as a growth factor) induces monomeric RTKs to form dimers.

  • Dimerization triggers autophosphorylation (cross-phosphorylation) of tyrosine residues located on the cytoplasmic catalytic domains of the receptor.

  • Phosphorylated tyrosines create high-affinity docking sites for cytosolic adapter proteins.

• Recruitment of Grb2 and SOS:

  • The cytosolic adapter protein Grb2 (growth factor receptor-bound protein 2) binds to the phosphorylated tyrosines of the activated RTK.

  • Grb2 subsequently recruits the guanine nucleotide exchange factor SOS (Son of Sevenless) to the inner leaflet of the plasma membrane.

• Activation of RAS:

  • RAS is a monomeric GTPase tethered to the cytoplasmic face of the plasma membrane via a covalently attached lipid anchor (a fatty acid tail).

  • In its resting basal state, RAS is bound to GDP and is inactive.

  • SOS acts as an exchange factor: it promotes the dissociation and release of GDP from RAS, allowing GTP to bind in its place.

  • This activation step is strictly a guanine nucleotide exchange reaction, not a direct phosphorylation of GDP to GTP.

  • Spatial compartmentalization prevents aberrant cell proliferation: SOS and downstream effectors are normally kept away from RAS until RTK activation recruits them to the membrane. Uncontrolled, constitutive RAS activation bypasses regulatory checks and drives oncogenesis.

• The Phosphorylation Cascade (RAF-MEK-ERK):

  • RAF (MAP Kinase Kinase Kinase / MAPKKK): Active RAS-GTP directly binds and recruits the serine/threonine kinase RAF. RAF undergoes autophosphorylation to achieve full catalytic activity.

  • MEK (MAP Kinase Kinase / MAPKK): Activated RAF hydrolyzes ATP to transfer a phosphate group onto MEK, converting MEK into its active phosphorylated state ($1\,\text{ATP}$ consumed per MEK phosphorylated).

  • ERK (MAP Kinase / MAPK): Active MEK transfers a phosphate group from ATP onto ERK, activating it.

  • Cascade Hierarchy and Nomenclature: ERK was identified first as MAP kinase (mitogen-activated protein kinase). The enzyme that phosphorylates it was designated MAP kinase kinase (MEK), and the upstream enzyme phosphorylating MEK was designated MAP kinase kinase kinase (RAF). A true phosphorylation cascade requires at least three sequentially acting kinases.

  • Cellular Response: Phosphorylated, active ERK translocates from the cytosol into the nucleus, where it phosphorylates specific transcription factors, driving the transcription of genes essential for cell growth and entry into mitosis.

G-Protein Coupled Receptors (GPCRs) and GTPase Molecular Switches

• Structural features of GPCRs:

  • Characterized by a polypeptide chain weaving through the membrane seven times via hydrophobic $\alpha$-helices.

  • Lacks intrinsic enzymatic activity; instead, its cytosolic loops associate with intracellular heterotrimeric G proteins.

• The Heterotrimeric G-Protein Cycle:

  • Heterotrimeric G proteins consist of three distinct subunits: $\alpha$, $\beta$, and $\gamma$.

  • The $\alpha$ subunit contains the guanine-nucleotide-binding pocket and intrinsic GTPase activity.

  • In the resting/basal state, the $\alpha$ subunit is bound to GDP and tightly associated with the $\beta\gamma$ complex, rendering the G protein inactive.

  • Ligand binding causes a conformational change in the GPCR, enabling it to act as a guanine nucleotide exchange factor for the $\alpha$ subunit.

  • The $\alpha$ subunit releases GDP and binds GTP; upon GTP binding, the $\alpha$ subunit undergoes a conformational switch and dissociates from both the GPCR and the $\beta\gamma$ dimer.

  • Both the GTP-bound $\alpha$ subunit and the free $\beta\gamma$ dimer can interact with downstream effector proteins (enzymes or ion channels).

  • Signal termination occurs when the $\alpha$ subunit hydrolyzes its bound GTP to GDP via its intrinsic GTPase activity:         $\text{GTP} \rightarrow \text{GDP} + \text{P}_i$

  • Once GDP-bound, the $\alpha$ subunit reassociates with the $\beta\gamma$ complex, resetting the system to the off state.

• Physiological Application: The Adrenergic Pathway:

  • Epinephrine (adrenaline) is synthesized and released by the adrenal glands situated atop the kidneys.

  • Epinephrine binds to cell-surface adrenergic receptors (GPCRs), initiating the fight-or-flight response.

  • The activated G protein stimulates downstream effectors that increase glycogen catabolism (glucose breakdown) and promote skeletal muscle contraction.

Monomeric vs. Heterotrimeric GTPases

• Monomeric GTPases (e.g., RAS):

  • Composed of a single protein unit anchored to the inner membrane by a lipid tail.

  • Requires a separate, specialized cytosolic guanine nucleotide exchange factor (SOS) that is recruited to the membrane to exchange GDP for GTP.

  • Direct downstream effector is RAF (MAPKKK).

• Heterotrimeric G Proteins (e.g., $\text{G}_\alpha\beta\gamma$):

  • Composed of three non-identical subunits ($\alpha$, $\beta$, $\gamma$).

  • The activated transmembrane GPCR itself serves as the exchange factor to trigger GDP dissociation and GTP binding on the $\alpha$ subunit.

  • Effector targets include enzymes such as adenylyl cyclase and phospholipase C, as well as membrane ion channels.

• Enzymatic Specificity:

  • GTPases are hydrolytic enzymes that cleave the terminal phosphoanhydride bond of GTP.

  • Because nucleotide reactivation relies on physical nucleotide exchange rather than direct phosphorylation of GDP, drugs or inhibitors targeting phosphate-transfer reactions (such as kinase inhibitors) do not directly block GTPase activation or cycling.

Intracellular Receptors and Direct Gene Regulation

• Mechanism of action:

  • Intracellular receptors reside within the interior of the cell (cytosol or nucleus) rather than spanning the plasma membrane.

  • Their ligands are small, hydrophobic, lipid-soluble molecules that diffuse freely through the phospholipid bilayer without requiring transport channels or surface receptors.

• The Glucocorticoid Receptor (GR) pathway:

  • Glucocorticoids are steroid hormones built upon a core structure of four fused hydrocarbon rings.

  • In the absence of hormone, GR is retained in the cytosol in an inactive state bound to inhibitory chaperone proteins.

  • Upon crossing the plasma membrane, the glucocorticoid ligand binds directly to GR, triggering the release of the inhibitory chaperones.

  • The activated ligand-receptor complex translocates into the nucleus, functioning directly as a transcription factor by binding specific regulatory DNA sequences.

  • Unlike cell-surface receptors, this pathway bypasses multi-step cytosolic signal transduction cascades, moving directly from reception to nuclear transcriptional response.

• Clinical relevance:

  • Glucocorticoids delivered via asthma inhalers diffuse into respiratory cells, bind GR, and repress the transcription of pro-inflammatory genes, thereby diminishing airway inflammation and restoring breathing.

Principles of Signal Transduction and Amplification

• Necessity for Transduction:

  • Large or polar (hydrophilic) signaling molecules cannot cross the hydrophobic lipid bilayer.

  • Signal transduction relays the information encoded by the extracellular ligand across the plasma membrane into intracellular target proteins.

• Key Functions of Multi-Step Transduction Cascades:

  • Signal Amplification: A single activated cell-surface receptor can activate multiple downstream G proteins or kinases; each kinase can phosphorylate multiple downstream substrates, generating an exponential cellular response from a minimal ligand concentration.

  • Signal Integration: Cells simultaneously encounter diverse extracellular cues; multi-step cascades allow cells to cross-talk, integrate competing inputs, and execute coordinated cellular behaviors.

  • Response Divergence: A single signaling molecule can trigger distinct physiological responses in different cell types by acting upon cell-specific suites of intracellular transducers and effector proteins.

• Kinases vs. Phosphatases:

  • Protein Kinases: Transfer the $\gamma$-phosphate group from ATP to specific target amino acid side chains (serine, threonine, or tyrosine).

  • Protein Phosphatases: Catalyze the hydrolytic removal of phosphate groups from proteins, returning them to their dephosphorylated state.

  • Phosphorylation acts as a reversible molecular switch: while it frequently activates target enzymes, it can also exert inhibitory effects or alter intracellular protein localization (e.g., exposing binding motifs that recruit partner proteins to the membrane).

Second Messenger Systems: The Cyclic AMP (cAMP) Pathway

• Characteristics of Second Messengers:

  • Small, non-protein, diffusible intracellular signaling molecules or ions (such as nucleotides, lipids, cyclic sugars, or ions).

  • Synthesized or released downstream of activated cell-surface receptors (primarily GPCRs, but also engaged by certain RTKs) to propagate and amplify signals throughout the cytoplasm.

• Synthesis and Degradation of Cyclic AMP:

  • Chemical Identity: Cyclic AMP (cAMP) is an RNA nucleotide derivative comprising an adenine base, a ribose sugar, and a cyclic monophosphate group.

  • Synthesis: Upon GPCR activation, the GTP-bound G protein $\alpha$ subunit activates the integral membrane effector enzyme adenylyl cyclase. Adenylyl cyclase cyclizes ATP into cyclic AMP ($\text{cAMP}$) with the release of pyrophosphate.

  • Degradation: The enzyme phosphodiesterase hydrolyzes the cyclic phosphodiester bond of $\text{cAMP}$, converting it into non-cyclic adenosine 5'-monophosphate (AMP) to terminate the signal.

• Downstream Target: Protein Kinase A (PKA):

  • Elevated cytosolic concentrations of $\text{cAMP}$ lead to the cooperative binding of $\text{cAMP}$ to the regulatory subunits of Protein Kinase A (PKA).

  • Binding releases the active catalytic subunits of PKA, which then phosphorylate downstream metabolic enzymes and regulatory proteins.

  • The primary functional outcome of active PKA is metabolic remodeling, accelerating catabolism to liberate cellular energy.

Second Messenger Systems: Phospholipase C, IP3, DAG, and Calcium Signaling

• Cleavage of Phosphatidylinositol 4,5-Bisphosphate ($\text{PIP}_2$):

  • A GPCR activated by ligand binding exchanges GDP for GTP on its coupled G protein.

  • The activated G protein stimulates the membrane-associated effector enzyme Phospholipase C (PLC).

  • Phospholipase C is a phosphodiesterase that cleaves the membrane phospholipid $\text{PIP}_2$ (phosphatidylinositol 4,5-bisphosphate) into two distinct second messengers:

    • Diacylglycerol (DAG): The hydrophobic lipid tail portion that remains embedded within the plasma membrane.

    • Inositol 1,4,5-Trisphosphate ($\text{IP}_3$): A hydrophilic phosphorylated inositol sugar head group containing three phosphate groups that readily dissociates from the membrane and diffuses freely through the cytosol.

• Signal Termination of $\text{IP}_3$:

  • Specific intracellular phosphatases hydrolyze phosphate groups from $\text{IP}_3$, converting it into inactive inositol derivatives that cannot activate downstream channels.

• Regulation and Mobilization of Calcium ($\text{Ca}^{2+}$):

  • Compartmental Concentration Gradients: Cytosolic $\text{Ca}^{2+}$ is maintained at extremely low concentrations under resting conditions. High concentrations of $\text{Ca}^{2+}$ are actively sequestered outside the cell in the extracellular fluid, as well as within internal intracellular organelles—specifically the Endoplasmic Reticulum (ER) and the mitochondria.

  • $\text{IP}_3$ Action on the ER: Soluble cytosolic $\text{IP}_3$ binds to specific ligand-gated $\text{Ca}^{2+}$ channels located on the ER membrane.

  • Binding of $\text{IP}_3$ opens these channels, triggering a rapid efflux of $\text{Ca}^{2+}$ out of the ER lumen and into the cytosol down its steep concentration gradient via facilitated diffusion.

  • Effector Activation: The sudden surge in cytosolic $\text{Ca}^{2+}$ activates a diverse array of calcium-responsive target proteins (e.g., calmodulin, protein kinase C, contractile proteins), directing physiological outcomes such as muscle contraction, secretion, and altered cellular metabolism.

Questions & Interactive Classroom Discussion

• Question: Can an ion channel permit the passage of ions across a membrane if it possesses only a single transmembrane helix?

  • Response: No. A single transmembrane $\alpha$-helix cannot form a central pore. An ion channel requires multiple transmembrane helices or multi-subunit arrangements spanning the membrane to assemble an internal hydrophilic passageway for ion diffusion.

• Question: What is the normal resting membrane potential of a cell, and what are the electrical consequences of cation versus anion transport?

  • Response: The interior of the cell is negatively charged relative to the outside at resting potential. Opening channels that permit the influx of cations makes the cell interior more positive, causing depolarization. Opening channels that permit the influx of anions makes the cell interior more negative, causing hyperpolarization.

• Question: Why is RAF formally designated as MAP kinase kinase kinase rather than simply because it is phosphorylated three times?

  • Response: The designation reflects its hierarchical position in the kinase cascade, not the number of phosphate groups it carries. ERK is the terminal MAP kinase (MAPK). MEK phosphorylates ERK, making MEK the MAP kinase kinase (MAPKK). RAF phosphorylates MEK, making RAF the kinase of the MAP kinase kinase—hence, MAP kinase kinase kinase (MAPKKK).

• Question: Does RAS transform into RAF, or does RAS activate RAF?

  • Response: RAS does not become RAF. RAS is a lipid-anchored monomeric GTPase that, when bound to GTP, physically recruits and allosterically activates the cytosolic protein kinase RAF.

• Question: During the kinase cascade, are phosphate groups transferred sequentially from one kinase directly onto the next?

  • Response: No. Activated kinases do not surrender their own phosphate groups to their downstream substrates. Instead, each active kinase utilizes a distinct molecule of ATP from the cytosol, transferring the $\gamma$-phosphate from that ATP onto its target kinase ($1\,\text{ATP}$ per phosphorylation event).

• Question: Where does the GTP come from during G-protein or RAS activation, and how does the G protein return to the off state?

  • Response: Activation occurs via nucleotide exchange, not phosphorylation of bound GDP. Under the influence of an activated receptor or exchange factor (SOS), the bound GDP dissociates entirely, and a new molecule of GTP from the cytosolic pool enters the nucleotide-binding pocket. Deactivation occurs because the G protein possesses intrinsic GTPase activity that autonomously hydrolyzes the bound GTP back into GDP and inorganic phosphate.

• Question: Do intracellular receptors undergo a multi-step cytoplasmic signal transduction relay?

  • Response: No. Because intracellular receptor ligands are hydrophobic steroids that diffuse directly across the membrane, the receptor itself acts as both the receptor and the ultimate effector. Once the ligand binds and inhibitory chaperone proteins dissociate, the complex translocates straight to the nucleus to alter transcription, bypassing multi-step cytoplasmic transduction cascades.