Introduction to Cell Signaling and G Protein-Coupled Receptors

Course Information and Final Exam Structure

• Final Exam Composition: These four final lectures on cell signaling will constitute approximately half ($50\%$) of the material on the final exam. The remaining half will cover older material from the rest of the course.

• Sequence: This is the first of three dedicated lectures on the topic of cell signaling.

Overview of the Cell Signaling Process

• General Components:

  • Ligand (Signaling Molecule): Produced and released by a specific signaling cell.

  • Transfer: The signal travels to the target cell via the bloodstream, the lymphatic system, or through the extracellular matrix (ECM).

  • Target Cell: This is defined as a cell that expresses the specific receptor required to bind the signaling molecule.

  • Receptor Activation: Ligand binding triggers a conformational change that activates the receptor.

  • Response: The target cell elicits a specific biological response.

  • Termination: The signaling process is eventually concluded through specific termination mechanisms.

• Seven Steps of Cell Surface Receptor Signaling:

  1. Synthesis of the signaling molecule.

  2. Release of the signaling molecule by the signaling cell (often via exocytotic/secretory vesicles).

  3. Transfer of the signaling molecule to the target cell.

  4. Binding of the signal to a specific receptor leading to activation.

  5. Initiation of one or more intracellular signal transduction pathways.

  6. Specific changes in cellular function, metabolism, or development.

  7. Termination of the cellular response.

• Cellular Outcomes: The effector proteins activated downstream can modulate:

  • Metabolism.

  • Cell function/movement.

  • Gene expression in the nucleus (common in developmental signals).

  • Negative feedback loops that diminish the activity of the activated receptor.

Types of Signaling and Stimuli

• Signaling Molecule Varieties:

  • Small Molecules: Acetylcholine ($ACh$) and glutamate (neurotransmitters).

  • Proteins/Peptides: Hormones and growth factors.

  • Membrane-bound/Tethered: Molecules attached to the plasma membrane of the signaling cell or embedded in the ECM.

  • Metabolites: Receptors can respond to oxygen or nutrients.

  • Physical Stimuli: Receptors for light, heat/cold, or touch.

• Classification of Extracellular Signaling:

  • Endocrine: Long-distance signaling where the hormone travels through the circulation (e.g., blood vessels) from glands like the thyroid, testes, or ovaries to distant target cells.

  • Paracrine: Short-distance signaling where the secretory cell and target cell are adjacent or in close proximity.

  • Autocrine: The signaling cell and target cell are the same; the cell secretes a signal that binds to its own surface receptors.

  • Plasma Membrane Attached Proteins: Signaling occurs through physical contact between the signal attached to the signaling cell membrane and the receptor on the target cell.

  • Intracrine Signaling: The signal is produced and binds to an intracellular receptor within the same cell without ever leaving the cell.

Classes of Receptors

• Plasma Membrane (Cell Surface) Receptors:

  • Bind to hydrophilic extracellular signaling molecules (small molecules, peptides, or proteins) that cannot cross the plasma membrane.

  • These include GPCRs, ion channel receptors, enzyme-coupled receptors, and those subject to regulated proteolysis or protein degradation.

• Intracellular (Cytosolic/Nuclear) Receptors:

  • Bind to hydrophobic molecules that passively diffuse through the plasma membrane.

  • Examples include steroid hormones (estrogen, progesterone, testosterone), retinoids, and thyroid hormones (thyroxine).

  • Upon binding, these often move to the nucleus to modify gene expression.

Receptor Properties and Cellular Responses

• Molecular Complementarity: Ligand-receptor binding is characterized by high affinity (often in the nanomolar or sub-nanomolar range). This tight binding depends on specific residues forming a pocket; growth hormone binding to the growth hormone receptor is a primary example.

• Differential Effects of a Single Signal: The same signal can cause different responses in different tissues (e.g., Acetylcholine affects skeletal muscle, heart muscle, and pancreatic glands differently). This occurs via two mechanisms:

  1. Cells express different types of receptors for the same ligand (different cytoplasmic domains but similar ligand-binding domains).

  2. Cells use the same receptor but activate different downstream effector molecules.

• Speed of Response:

  • Fast: Changes in activity/function of proteins already present in the cell (e.g., GPCR pathways).

  • Slow: Changes in the amount of specific proteins via gene expression (transcription of $DNA$ to $RNA$) or protein stability.

  • Ion Flux: Rapid changes in intracellular ion concentrations.

Conserved Components: Second Messengers

Second messengers are low molecular weight, intracellular signaling molecules with short half-lives that increase or decrease in concentration upon receptor activation.

• Cyclic AMP (cAMP): Adenosine monophosphate with a covalent bond between the phosphate and the $3'\,OH$ of the sugar. It activates Protein Kinase A (PKA).

• Cyclic GMP (cGMP): Similar to cAMP but with guanine. It activates Protein Kinase G (PKG).

• 1,2-diacylglycerol (DAG): Consists of a glycerol molecule with two fatty acid groups. It activates Protein Kinase C (PKC).

• Inositol 1,4,5-triphosphate (IP3): An inositol molecule with three phosphates. It opens $Ca^{2+}$ channels in the endoplasmic reticulum (ER).

• Calcium Ions ($Ca^{2+}$): Acts as a messenger by binding to proteins like Calmodulin.

  • Calmodulin: Contains four EF hands (helix-loop-helix motifs). Upon binding four $Ca^{2+}$ ions, it undergoes a conformational change from a stretched to a circular orientation to bind target proteins.

Conserved Components: GTP-Binding Proteins (GTPases)

GTPases act as molecular switches: active when bound to $GTP$, inactive when bound to $GDP$.

• Regulatory Proteins:

  • GEF (Guanine nucleotide Exchange Factor): Exchanges $GDP$ for $GTP$ to turn the protein "on."

  • GAP (GTPase Activating Protein): Stimulates $GTP$ hydrolysis to turn the protein "off."

• Major Classes:

  • Heterotrimeric G Proteins: Consist of $\alpha$, $\beta$, and $\gamma$ subunits. The $G\alpha$ subunit binds $GTP/GDP$ and is the primary transducer.

  • Monomeric (Small) GTPases: Includes the RAS superfamily (e.g., RAS, RAN, RAC, Rho). RAS is linked to receptors indirectly through adapter proteins.

• Experimental Measurement (Pull-down Assay):

  • Mechanism: Uses beads (e.g., $PBD\,agarose$) coated with a protein like PAC-1, which only binds to the active form (e.g., $RAC-GTP$).

  • Process: Lysates are incubated with beads and centrifuged. The pellet is run on a gel and analyzed via immunoblotting with an anti-RAC antibody.

  • Example: Treatment of stem cells with Platelet Derived Growth Factor (PDGF) leads to rapid RAC activation within $1\,minute$.

Conserved Components: Kinases and Phosphatases

• Protein Kinases: Add phosphate groups (phosphorylation) using $ATP$. Can activate or inactivate a protein.

• Protein Phosphatases: Remove phosphate groups (dephosphorylation).

• MAP Kinase (MAPK) Pathway Example:

  1. Activated receptor triggers RAS-GTP.

  2. RAS-GTP activates MAP Kinase Kinase Kinase (MAPK3/RAF).

  3. RAF phosphorylates MAP Kinase Kinase (MAPK2/MEK).

  4. MEK phosphorylates MAP Kinase (MAPK/ERK).

  5. ERK phosphorylates transcription factors to alter gene expression.

G Protein-Coupled Receptors (GPCRs) and Trimeric G Proteins

• GPCR Structure: Integral membrane proteins with seven ($7$) membrane-spanning alpha-helices. The N-terminus is exoplasmic, and the C-terminus is cytosolic. Loop $C3$ is typically the largest intracellular loop.

• Heterotrimeric G Protein Cycle:

  1. Ligand binds GPCR; receptor undergoes conformational change.

  2. Activated GPCR binds to $G\alpha$ (part of the \alphạ̇\beta\gamma complex anchored to the membrane by fatty acid modifications).

  3. $G\alpha$ releases $GDP$ and binds $GTP$.

  4. $G\alpha-GTP$ dissociates from the $\beta\gamma$ dimer and the receptor.

  5. Active $G\alpha-GTP$ (and sometimes $\beta\gamma$) activates an effector protein.

  6. $G\alpha$ hydrolyzes $GTP$ to $GDP$, dissociates from the effector, and re-associates with $\beta\gamma$.

• Classes of G-Alpha Subunits:

  • $G\alpha_{s}$ (Stimulatory): Activates adenylyl cyclase; increases cAMP. (e.g., beta-adrenergic receptors for epinephrine, glucagon).

  • $G\alpha_{i}$ (Inhibitory): Inhibits adenylyl cyclase; decreases cAMP. (e.g., alpha-2 adrenergic receptors).

  • $G\alpha_{olf}$ (Olfactory): Activates adenylyl cyclase; increases cAMP for smell sensing.

  • $G\alpha_{q}$ and $G\alpha_{o}$: Activate Phospholipase C (PLC); increase IP3 and DAG levels. (e.g., acetylcholine receptors).

  • $G\alpha_{t}$ (Transducin): Activates cyclic GMP phosphodiesterase; decreases cGMP. (Used in Rhodopsin for light sensing).

Specific Case Study: Epinephrine Signaling

• Epinephrine (Adrenaline): Regulates the "fight or flight" response.

• Tissue-Specific Effects:

  • Cardiac muscle: Increased contraction.

  • Skeletal muscle/Liver: Breakdown of glycogen to glucose for energy.

  • Smooth muscle: Intestinal relaxation; arterial constriction.

  • Adipose tissue: Breakdown of fats to fatty acids.

• Pathway: Binds beta-adrenergic receptors $→ G\alpha_{s} →$ activation of Adenylyl Cyclase.

• Adenylyl Cyclase Structure: Membrane-bound enzyme with two clusters of six transmembrane domains and two catalytic domains on the cytoplasmic face. It converts $ATP$ to $cAMP$ by releasing two phosphates ($PP_{i}$) and forming a cyclic bond with the remaining phosphate.