🚦 Lecture 10: Signal Transduction + GPCR (G Protein-Coupled Receptors)

Signal Transduction – How Cells Sense External Signals

Overview
 Transcription factors respond to extracellular signals from hormones and signaling peptides
 Signal transduction connects external signals to changes in gene expression and metabolism

Hydrophilic Hormones
 Cannot cross the cell membrane
 Bind to plasma membrane receptors
 Activate signal transduction pathways:
  • Some receptors activate G-proteins → trigger various intracellular pathways or generate second messengers
  • Tyrosine kinase receptors → sequential activation of downstream kinases

Hydrophobic Hormones
 Diffuse through the cell membrane
 Bind cytosolic receptors directly

Outcome
 Both types of hormones initiate signal transduction
 Lead to changes in gene expression and cellular metabolism

Signal Transduction Pathway – Hydrophilic Hormones *Don’t need to know

Step 1 – Ligand Binding
 A receptor protein (R) on the plasma membrane binds a hydrophilic hormone (H)
 Binding triggers a conformational change in the receptor

Step 2 – Receptor Activation
 Activated receptor interacts with a signal transduction protein
 Often a GTP-binding protein, kinase, or phosphatase

Step 3 – Signal Cascade
 Signal transduction protein activates or inhibits other downstream signaling proteins
 One protein can activate multiple downstream targets → signal amplification

Step 4 – Further Signaling
 Activated proteins continue to transmit the signal through the pathway
 Amplifies and diversifies the cellular response

Step 5 – Effector Activation
 Some signaling proteins activate effector proteins (E)
 Effectors can be enzymes, transcription factors, transport proteins, or ion channels
 Effectors carry out the cellular response

Step 6 – Feedback Control
 Proteins in the pathway or effectors can modify the receptor or early signaling proteins
 Inhibits or blocks early steps in the pathway
 Can trigger receptor degradation → reduces receptor numbers → lowers cell sensitivity to the ligand

Key Points
 Signal amplification ensures a small number of ligands produces a large cellular response
 Feedback mechanisms maintain control and prevent overstimulation

Cell-to-Cell Signaling – Types and Mechanisms

(a) Endocrine Signaling
 Hormones are secreted by endocrine glands into the blood
 Travel long distances to reach distant target cells
 Etymology: endo- = inside, crine = secrete → “secrete inside” (into the blood)

(b) Paracrine Signaling
 Signaling molecules are released by a cell to act on nearby adjacent cells
 Short-distance signaling
 Etymology: para- = beside/near → “secrete beside” (affects neighboring cells)

(c) Autocrine Signaling
 A cell secretes signaling molecules that act on itself
 Can regulate its own activity or gene expression
 Etymology: auto- = self → “self-secretion” (acts on the same cell)

(d) Signaling by Plasma-Membrane-Attached Proteins
 Proteins on the plasma membrane of one cell interact directly with receptors on an adjacent cell
 Does not require secretion of molecules
 Allows direct cell-to-cell communication

Key Points
 Signaling distance varies: autocrine/paracrine = micrometers, endocrine = meters

Overview of Cell Signaling – Hormones and Receptors

Signal Reception
 All cells respond to extracellular signals or stimuli
 Signals activate plasma membrane receptors or cytosolic receptors

Receptor Function
 Activated receptors can:
  • Act as transcription factors → change gene expression
  • Activate G-protein switches → regulate multiple downstream pathways
  • Generate intracellular second messengers → amplify and transmit the signal

Signal Regulation
 Protein activity is controlled by:
  • Phosphorylation by kinases
  • Dephosphorylation by phosphatases
 These modifications regulate signaling pathways and can amplify intracellular signals

Overview of Cell Signaling – Hydrophobic vs Hydrophilic Signals

Hydrophobic Signals
 Examples: steroids, retinoids, thyroxine

Steps:

1. Signal molecule diffuses through the plasma membrane

2. Binds to a cytosolic receptor → forms receptor-signal complex

3. Complex moves into the nucleus

4. Binds DNA transcription-control regions → activates or represses gene expression

Hydrophilic Signals
 Examples: small molecules (adrenaline, acetylcholine), peptides (glucagon, yeast mating factors), proteins (insulin, growth hormone)

Steps:

1. Signal binds to an inactive cell-surface receptor (4)

2. Receptor undergoes conformational change → becomes active (4)

3. Activated receptor activates downstream signal transduction proteins or generates second messengers (5)

4. Signal transduction activates effector proteins (6)

5. a. Effector proteins stay in cytosol → modify enzymes → short-term changes in metabolism, movement, or function (7a)

b. Effector proteins enter the nucleus → long-term changes in gene expression (7b)

Termination / Down-Modulation:
6. Negative feedback from intracellular signalling molecules (8)
7. Removal of the extracellular signal (9)

Key Points:
 Hydrophobic → cytosol receptor → nucleus → gene expression modification
 Hydrophilic → membrane receptor → signal transduction → effector → cytosol or nucleus
 Signal amplification and feedback control regulate strength and duration of response

Hydrophilic Signaling – Stepwise Pathway

Steps:
4. Signal molecule binds to a specific cell-surface receptor → receptor undergoes conformational change → receptor becomes active
5. Activated receptor triggers downstream signal transduction proteins and/or generates second messengers
6. Signal transduction activates effector protein(s)
7a. Short-term response: cytosolic effectors modify enzymes → changes in metabolism, function, or movement
7b. Long-term response: effectors move into the nucleus → changes in gene expression
8. Termination / Down-regulation: negative feedback from intracellular signaling molecules inhibits early steps and/or:
9. Removal of the extracellular signal from the receptor ends the pathway

Key Points:
 Hydrophilic signals cannot cross the membrane → require membrane receptors
 Pathway can amplify the signal through multiple downstream proteins
 Feedback ensures the response is controlled and reversible

Growth Hormone Binding to Two Receptors (Steps from textbook, don’t need to know)

Overview
 A single growth hormone (GH) protein binds simultaneously to two growth hormone receptors (GHRs)
 Binding is mediated by multiple weak, noncovalent forces

Key Findings from Structural Studies

• GH-Receptor Interface: 28 amino acids on GH interact with the first receptor

• Critical Residues: Only 8 amino acids on GH contribute ~85% of the binding energy
   • These 8 residues are distant in the primary sequence but adjacent in the folded 3D structure

• Receptor Contribution: Two tryptophan residues on the receptor provide most of the binding energy
   • Other receptor amino acids at the interface also contribute

Binding Sequence

1. GH binds to the first receptor molecule (involving GH’s key residues and receptor interface residues)

2. A second receptor molecule binds on the opposite side of GH
    • Uses the same critical amino acids on the receptor
    • Interacts with different residues on GH compared to the first receptor

Key Points
 - GH binding involves specific residues on both hormone and receptor
 - Multiple weak interactions combine to create strong and specific binding
 - Protein folding brings distant amino acids close together → essential for receptor interaction

Hormone Receptors – Activation of Effector Proteins

Overview
 Receptors sense hormones and activate effector proteins inside the cell

Effector Proteins
 Some are enzymes that make second messengers → carry and amplify the signal
 Some are kinases or phosphatases → modify downstream proteins by adding or removing phosphate groups
 Some are G-protein switches → directly activate downstream proteins

Outcome
 Receptors transmit the hormone signal and control cellular responses like metabolism, function, or gene expression

Intracellular Second Messengers

Overview
 Second messengers are small molecules produced by effector proteins
 They transmit and amplify signals from hormone-receptor complexes

Properties
 Small and short-lived
 Diffuse rapidly within the cell
 Enable enzymatic amplification of the signal

Types
 Water-soluble: Ca²⁺ ions, cAMP, cGMP → move freely in cytosol
 Lipid-soluble: DAG, IP₃ → can interact with membrane-associated proteins

Outcome
 Carry the signal from activated receptors to other proteins, amplifying the cellular response

Signal Transduction by Protein Phosphorylation

Overview
 Proteins can be modified by multiple protein kinases or phosphatases
 These modifications regulate protein activity and propagate signals in the cell

Regulation of Kinases and Phosphatases
 Activity can be controlled by:
  Phosphorylation of the kinase or phosphatase itself
  Binding to other proteins
  Binding of second messenger molecules

Effects of Phosphorylation
 Phosphorylation can activate or inhibit a protein’s function
 Specific protein phosphatases remove phosphate groups to reverse kinase effects

Outcome
 Phosphorylation and dephosphorylation allow fine control of signaling pathways and amplify cellular responses

Protein Kinases and Phosphatases in Animal Cells

Kinase Types
 Two main types of protein kinases in animal cells:
  Tyrosine (Y) kinases
  Serine (S) / Threonine (T) kinases

Human Genome
 Encodes more than 600 different protein kinases (~1.7% of all genes)
 Encodes over 200 different protein phosphatases

Overview
 Kinases add phosphate groups to proteins, regulating their activity
 Phosphatases remove phosphate groups, reversing kinase effects
 Together, they control cellular signaling pathways precisely

Activation of Protein Kinase A (PKA) by Phosphorylation *Don’t need to know

Overview
 Protein kinase A (PKA) exists in an inactive, unphosphorylated form
 Activation occurs through phosphorylation of a threonine residue (Thr197) in the activation loop

Mechanism
 Phosphorylation at Thr197 causes a conformational change in the activation loop
 This change allows PKA to bind ATP and substrate proteins, enabling its kinase activity
 Phosphorylation also allows PKA to bind its inhibitory subunit R, which regulates activity via cAMP

Additional Notes
 PKA can autophosphorylate Thr197
 Other cellular kinases can also phosphorylate this residue
 Similar phosphorylation-dependent activation occurs in many other protein kinases

Outcome
 Phosphorylation of Thr197 switches PKA from inactive to active
 Enables it to phosphorylate downstream target proteins and propagate cellular signals

G-Proteins: GTPase Switch Proteins

Overview
 Hydrophilic hormones bind to membrane receptors, often called G-protein coupled receptors (GPCRs)
 These receptors activate G-proteins, which are GTPase switch proteins

Activation Mechanism
 G-proteins exchange GDP for GTP, causing a conformational change and activation
 Active G-proteins transmit signals to downstream effectors

Inactivation
 G-proteins have intrinsic GTPase activity
 GTPase-Accelerating Proteins (GAPs) can speed up GTP hydrolysis
 Hydrolysis of GTP to GDP inactivates the G-protein and stops the signal

Outcome
 G-proteins act as molecular switches, turning signaling on when bound to GTP and off when hydrolyzing it to GDP

G-Proteins: GTPase Switch Proteins – Regulation

Overview
 G-protein activity is controlled by the exchange of GDP for GTP

Activation
 Binding of GTP switches the G-protein ON
 Activator proteins called GEFs (Guanine nucleotide exchange factors) promote this GDP → GTP exchange
 Active G-proteins transmit signals to downstream effectors

Inactivation
 After signaling, GTP is hydrolyzed to GDP → switches the G-protein OFF
 Inactivator proteins such as GAPs (GTPase-activating proteins) or RGS (regulators of G-protein signaling) accelerate this hydrolysis

Outcome
 G-proteins act as molecular switches, cycling between active (GTP-bound) and inactive (GDP-bound) states to regulate signal transduction

Switching Mechanism of Monomeric G-Proteins

Overview
 G-protein conformation changes depending on whether GDP or GTP is bound

Inactive State
 G-protein bound to GDP is inactive
 Can interact with upstream activator proteins

Active State
 G-protein bound to GTP is active
 Can interact with downstream effector proteins, often enzymes
 Effectors produce second messenger molecules to transmit and amplify the signal

Key Point
 The switch from GDP to GTP is not a phosphorylation event
 It is a conformational change caused by nucleotide exchange

Major Classes of Mammalian Heterotrimeric G Proteins and Their Effectors *Just know if second messengers increase or decrease

Gαs
 Effector: Adenylyl cyclase
 Second Messenger: cAMP (increased)
 Receptor Examples: β-Adrenergic (epinephrine) receptor, glucagon, serotonin, vasopressin receptors

Gαi
 Effector: Adenylyl cyclase, K⁺ channels (Gβγ activates effector)
 Second Messenger: cAMP (decreased), change in membrane potential
 Receptor Examples: α2-Adrenergic receptor, muscarinic acetylcholine receptor

Gαolf
 Effector: Adenylyl cyclase
 Second Messenger: cAMP (increased)
 Receptor Examples: Odorant receptors in the nose

Gαq
 Effector: Phospholipase C
 Second Messenger: IP₃, DAG (increased)
 Receptor Examples: α1-Adrenergic receptor

Gαo
 Effector: Phospholipase C
 Second Messenger: IP₃, DAG (increased)
 Receptor Examples: Acetylcholine receptor in endothelial cells

Gαt
 Effector: cGMP phosphodiesterase
 Second Messenger: cGMP (decreased)
 Receptor Examples: Rhodopsin (light receptor) in rod cells

Generation and Breakdown of cAMP *Don’t need to know

Overview
 cAMP (cyclic AMP) is a second messenger produced from ATP by the enzyme adenylyl cyclase

Generation
 ATP is converted into cAMP by adenylyl cyclase when a G-protein (Gαs) activates it
 cAMP carries and amplifies the signal to downstream effectors

Breakdown
 cAMP phosphodiesterase converts cAMP into AMP, terminating the signal
 This ensures that the signal is short-lived and tightly controlled

Outcome
 cAMP levels regulate cellular responses by activating cAMP-dependent protein kinases (PKA) and other effectors

Transduction and Amplification of Signals

Overview
 Second messengers, like cAMP, transmit and amplify signals from hormones

Example: Epinephrine Signaling
 Epinephrine binds to its receptor → activates adenylyl cyclase → produces cAMP
 cAMP activates Protein Kinase A (PKA) → PKA activates multiple downstream enzymes

Amplification
 One epinephrine molecule (10⁻¹⁰ M) → many cAMP molecules (10⁻⁶ M)
 Each PKA molecule can activate several enzymes → large-scale production of final products

Outcome
 Signal amplification ensures that a small amount of hormone can trigger a strong cellular response