BMB:3110 - Signal-Transduction Pathways

Chapter 13: Signal Transduction Pathways

Signal transduction pathways are molecular circuits that involve a similar multi-step process for information transfer across different pathways.

Basic Steps in Signal Transduction

Primary Messenger: This is a stimulus or trigger that releases the signal molecule. Examples include hormones, neurotransmitters, or touch.
Reception of Primary Messenger: Receptors, which are typically integral membrane proteins, bind the primary messenger extracellularly and transfer information across the cell membrane to the intracellular environment. This binding often induces a conformational change in the receptor.
Transduction and Amplification: Structural changes in the receptor lead to increased local concentrations of second messengers (like cAMP, Ca$^{2+}$, DAG, IP$_{3}$) that relay information from the receptor to downstream targets. This step often involves protein-protein interactions and protein modifications (e.g., phosphorylation). A single activated receptor can activate multiple downstream molecules, leading to significant signal amplification.
Physiological Response: This involves the activation or inhibition of final effectors. For example, enzymes that control metabolic pathways can be activated or gene expression can be altered.
Termination of Signaling: Signaling must be stopped (downregulated) promptly after the information has been transferred to ensure precise control and to allow the cell to respond to new or changing signals. Mechanisms include ligand dissociation, enzyme activity (e.g., GTP hydrolysis, dephosphorylation), and degradation of second messengers.

Overview of Receptors and Transducers

Cellular signaling uses a relatively small number of conserved structures and mechanisms.

Receptor Proteins: Bind to extracellular molecules and transmit information across the membrane to the intracellular space.
- 7 Transmembrane-Helix Receptors (7TM) / G-Protein Coupled Receptors (GPCRs): Example: ext{β2}-adrenergic receptor, which binds epinephrine.
- Dimeric Receptors that Recruit Kinases: Example: Growth hormone (GH) receptor.
- Dimeric Receptor Kinases: Example: Epidermal growth factor receptor (EGFR).
Transducers: Components that produce second messengers and/or mediate protein-protein interactions, acting in cascades to regulate and amplify signaling.
- Trimeric G-protein, adenylate cyclase, protein kinase A (PKA).
- Phospholipase C (PLC), protein kinase C (PKC).
- Janus kinase (JAK).
- Grb-2, Sos, Ras (a monomeric G protein/small GTPase).
- IRS, PI3 kinase, PIP3-dependent kinase (PDK1), Akt kinase.

7TM Receptors (GPCRs)

Structure: Characterized by seven transmembrane helices that span the plasma membrane.
Signal Versatility: They receive a wide variety of signals, including hormones, odorants, neurotransmitters, and photons.
Mechanism: Ligand binding to the extracellular domain causes a specific structural (conformational) change in the cytoplasmic domain of the receptor.
Example: The ext{β2}-adrenergic receptor recognizes epinephrine (adrenaline) and is crucial for initiating the "fight or flight" response.
Biological Functions: 7TM receptors mediate diverse biological functions:
- Hormone secretion, neurotransmission, chemotaxis, exocytosis.
- Control of blood pressure, cell growth and differentiation.
- Sensory perceptions: Smell, taste, vision.
- Viral infection/entry into a host.
Disease Relevance: Dysfunctional 7TM receptors are linked to various diseases and conditions:
- Color blindness, familial hypogonadism, short stature (mutated growth hormone receptor), extreme obesity, congenital hypothyroidism, Hirschsprung disease, precocious puberty, night blindness.
- Approximately 50\% of all pharmaceutical drugs in use target 7TM receptors.

G-Protein Activation by GPCRs

Ligand Binding: Epinephrine binding to the extracellular domain of the ext{β2}-adrenergic receptor induces a conformational change on its cytoplasmic side.
G-Protein Activation: This conformational change activates a trimeric G-protein (heterotrimer composed of ext{α}, ext{β}, and ext{γ} subunits) associated with the receptor.
GDP/GTP Exchange: The activated GPCR functions as a Guanine-nucleotide Exchange Factor (GEF), causing a conformational change in the G ext{α} subunit, which facilitates the exchange of bound GDP for GTP.
Dissociation: When GTP binds to G ext{α}, the G ext{α} subunit dissociates from the G ext{βγ} dimer. Both the GTP-bound G ext{α} and the G ext{βγ} dimer can then interact with and activate downstream effector proteins.
Signal Amplification: One activated receptor can activate many G ext{α} subunits.
GTP Hydrolysis: G ext{α} inherently possesses slow GTPase enzymatic activity, which eventually hydrolyzes GTP back to GDP + ext{Pi}. This returns G ext{α} to its inactive, GDP-bound state, allowing it to reassociate with G ext{βγ}.

Regulation of G-Protein Activity

GEFs (Guanine-nucleotide Exchange Factors): Catalyze the exchange of GDP for GTP, thereby activating G-proteins. Activated GPCRs act as GEFs for their associated G-proteins.
GAPs (GTPase Activating Proteins): Stimulate the inherent GTPase activity of the G ext{α} subunit, accelerating GTP hydrolysis to GDP + ext{Pi}. This inactivates the G-protein.

Adenylate Cyclase Activation and cAMP Production

Activated, GTP-bound G ext{α} dissociates from G ext{βγ} and directly binds to and activates the membrane-bound enzyme adenylate cyclase.
Adenylate cyclase catalyzes the conversion of ATP to cyclic AMP (cAMP), a crucial second messenger.
- ext{ATP} ightarrow ext{cAMP} + ext{PP}_ ext{i}
Signal Amplification: One active adenylate cyclase enzyme can produce multiple cAMP molecules.

Protein Kinase A (PKA) Activation

cAMP is the primary activator of Protein Kinase A (PKA).
PKA, once activated, phosphorylates specific serine and threonine residues on various target proteins, altering their activity.
Downstream Targets/Responses: Secretion of digestive fluids in the stomach, intestines, and pancreas; dispersion of melanin pigment granules in skin; reduction of platelet aggregation.

Resetting the 7TM Pathway

Ligand Dissociation: The primary messenger (ligand) dissociates from the receptor, causing the receptor to return to its inactive conformation.
GTP Hydrolysis: The intrinsic GTPase activity of G ext{α} hydrolyzes its bound GTP to GDP, leading to the reassociation of inactive G ext{α} with G ext{βγ}.

Olfaction (Sense of Smell) - A GPCR Example

Each olfactory neuron expresses one specific odorant receptor (GPCR).
Activation of the GPCR by a specific odorant is highly specific.
The downstream signaling pathway within olfactory neurons is conserved, involving G-protein activation, adenylate cyclase, and cAMP production, ultimately leading to an action potential.

Other Second Messengers: The Phosphoinositide Cascade

This pathway involves three key second messengers: PIP${2}$, DAG, and IP${3}$.

PIP$_{2}$ Cleavage: Activated receptor (often a GPCR) activates Phospholipase C (PLC).

PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP$_{2}$) into two second messengers:
- Diacylglycerol (DAG): Remains embedded in the membrane.
- Inositol 1,4,5-trisphosphate (IP$_{3}$): A water-soluble molecule that diffuses into the cytoplasm.

IP${3}$ Effects: IP${3}$ binds to a ligand-gated ion channel on the endoplasmic reticulum (ER), causing the rapid release of intracellular Ca$^{2+}$ stores into the cytoplasm.
DAG and Ca$^{2+}$ Effects: Both DAG (in the membrane) and the released Ca$^{2+}$ (in the cytoplasm) act in concert to activate Protein Kinase C (PKC). PKC then phosphorylates various target proteins.
Signal Termination: IP$_{3}$ is rapidly degraded (within seconds) to terminate the signal.

Ca$^{2+}$ as a Versatile Intracellular Messenger

Regulation: Intracellular Ca$^{2+}$ concentration is tightly regulated, with resting cytoplasmic levels kept low.
Calmodulin: This ubiquitous protein binds almost all Ca$^{2+}$ ions in cells.
- Structure: Calmodulin contains four "EF hands," each capable of binding one Ca$^{2+}$ ion.
- Activation: Calmodulin is activated when Ca$^{2+}$ stores are released into the cytoplasm.
Ca$^{2+}$ Signaling: Activated calmodulin-Ca$^{2+}$ complex binds to and regulates various target proteins, including the calmodulin-dependent protein kinase (CaM kinase), which phosphorylates other target proteins.
Roles: CaM kinases regulate metabolism, ionic permeability, and neurotransmitter synthesis and release.
Restoration: The Ca$^{2+}$-ATPase pump is stimulated by calmodulin-Ca$^{2+}$ complex to pump Ca$^{2+}$ out of the cytoplasm or into the ER, thereby restoring low intracellular Ca$^{2+}$ levels and terminating the signal.

Dimerized Receptors: Receptor Kinases

These receptors dimerize upon ligand binding, leading to activation of intrinsic or associated kinase activity.

Dimeric Receptor Kinases: Epidermal Growth Factor Receptor (EGFR)

Mechanism: The binding of Epidermal Growth Factor (EGF) to EGFR causes the receptor monomers to dimerize.
Cross-Phosphorylation: Dimerization brings the cytoplasmic kinase domains into proximity, leading to cross-phosphorylation (autophosphorylation) of tyrosine residues on the opposing receptor monomer.
Adaptor Protein Binding: The phosphorylated tyrosines serve as binding sites for adaptor proteins, such as Grb-2.
Ras Activation: Grb-2 recruits Sos, which acts as a GEF (Guanine-nucleotide Exchange Factor) for Ras. Ras is a small G protein (small GTPase).
Downstream Effects: Activated Ras, by exchanging GDP for GTP, initiates a phosphorylation cascade that ultimately promotes cell growth and proliferation.

Small GTPases

Regulation: Small GTPases (like Ras) are regulated similarly to trimeric G proteins:
- They are active when bound to GTP and inactive when bound to GDP.
- GEFs (e.g., Sos for Ras) promote the exchange of GDP for GTP, activating them.
- They have slow intrinsic GTPase activity. GAPs (GTPase Activating Proteins) accelerate GTP hydrolysis, thus inactivating them.
Subfamilies and Functions:
- Ras: Regulates cell growth through serine or threonine protein kinases.
- Rho: Reorganizes the cytoskeleton through serine or threonine protein kinases.
- Arf: Activates the ADP-ribosyltransferase of the cholera toxin A subunit; regulates vesicular trafficking pathways; activates phospholipase D.
- Rab: Key role in secretory and endocytotic pathways.
- Ran: Functions in the transport of cargo into and out of the nucleus.

Dimeric Receptors that Recruit Kinases: Growth Hormone (GH) Receptor

Mechanism: The GH receptor dimerizes upon binding of a single growth hormone ligand molecule.
Kinase Recruitment: Unlike EGFR, the GH receptor itself does not have intrinsic kinase activity. Instead, it is constitutively associated with separate tyrosine kinases.
Associated Kinase: The GH receptor is associated with Janus Kinase 2 (JAK2).
Cross-Phosphorylation and Activation: Upon receptor dimerization, the associated JAK2 molecules are brought into proximity and cross-phosphorylate each other, leading to their activation.
Downstream Signaling: Activated JAK2 phosphorylates specific tyrosine residues on the GH receptor, which then serve as binding sites for other signaling proteins, such as STAT5. Activated JAK2 also phosphorylates STAT5, which then dimerizes, translocates to the nucleus, and regulates gene expression.

Signaling in Metabolism: The Insulin Receptor

Type: The insulin receptor is a receptor tyrosine kinase.
Activation: Insulin binding leads to cross-phosphorylation (autophosphorylation) of tyrosine residues on the intracellular domains of the insulin receptor. This activates the receptor's intrinsic tyrosine kinase activity.
IRS Phosphorylation: The activated insulin receptor tyrosine kinase phosphorylates Insulin Receptor Substrates (IRS) proteins on their tyrosine residues.
PI3K Activation: Phosphorylated IRS proteins then recruit and activate Phosphoinositide 3-kinase (PI3K).
PIP${2}$ to PIP${3}$ Conversion: PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP${2}$) in the membrane to form phosphatidylinositol 3,4,5-trisphosphate (PIP${3}$). This step represents enzymatic amplification.
PDK1 Activation: PIP$_{3}$ recruits and activates PDK1 (PIP3-dependent protein kinase 1).
Akt Activation: PDK1 then phosphorylates and activates Akt protein kinase (also known as Protein Kinase B, PKB). This is another enzymatic amplification step.
Physiological Response: Activated Akt promotes the translocation of glucose transporters (specifically GLUT4) to the cell surface, increasing cellular glucose uptake. It also regulates various metabolic processes like glycogen synthesis and protein synthesis.

Dysfunctional Signal Transduction and Disease

Disruptions in signal transduction pathways can have severe consequences, contributing to various diseases, including bacterial infections and cancer.

Bacterial Toxins Affecting G-Proteins

Cholera Toxin: Produced by Vibrio cholerae. This toxin modifies the G ext{α} subunit, preventing its intrinsic GTPase activity. This results in the stabilization of the GTP-bound, active form of G ext{α}. Continuously active G ext{α} leads to sustained activation of adenylate cyclase and high cAMP levels, causing PKA to remain active and open chloride channels. This results in excessive loss of NaCl and water from intestinal cells, leading to severe diarrhea and dehydration.
Whooping Cough Toxin: Produced by Bordetella pertussis. This toxin weakens the affinity of G ext{α} for GTP, effectively keeping G ext{α} in its inactive, GDP-bound state. This also affects ion permeability in respiratory cells.

Dysfunctional Signal Transduction and Cancer

Cancer: Characterized by unregulated cell growth, which often stems from defects in signal transduction pathways that control cell division and survival.
Oncogenes: Mutations in genes encoding signal transduction proteins can contribute to tumorigenesis (formation of tumors).
- Proto-oncogenes: Normal genes that, when mutated, become oncogenes (cancer-causing genes).
- EGFR Overexpression: Overexpression or constitutive activation of the Epidermal Growth Factor Receptor (EGFR) can lead to uncontrolled cell division.
- Ras Mutations: Mutations in the Ras gene are found in approximately 25\% of all human tumors, and up to 90\% in certain cancers (e.g., pancreatic cancer). Common mutations (e.g., at Glycine 12 (G12), Glycine 13 (G13), or Glutamine 61 (Q61)) prevent Ras from hydrolyzing GTP, trapping it in its constitutively active, GTP-bound state, continuously promoting cell growth.
Tumor Suppressor Genes: These genes normally repress cell growth and proliferation. If both copies of a tumor suppressor gene are mutated or lost, cancer can result. Some tumor suppressor genes encode phosphatases that downregulate signal transduction pathways.
Chronic Myelogenous Leukemia (CML):
- Approximately 90\% of CML patients have a specific chromosomal translocation that fuses the c-abl gene (a tyrosine kinase) with the bcr gene, creating the Bcr-Abl fusion protein.
- This fusion results in a constitutively active kinase that drives uncontrolled cell proliferation.
- Understanding this specific signaling defect led to the development of targeted therapies like Gleevec (Imatinib), which effectively inhibits the Bcr-Abl kinase, demonstrating how knowledge of signal transduction pathways can lead to better cancer treatments.

Key Concepts for Review

The common steps involved in signal transduction pathways: primary messenger, reception, transduction, amplification, response, and termination.
The definition and function of transducers and second messengers.
Mechanisms by which different receptor classes transmit information: 7TM receptors/GPCRs, dimeric receptor kinases (e.g., EGFR), and dimeric receptors that recruit kinases (e.g., GH receptor).
The workings of G proteins and small GTPases (GTP/GDP cycling, activity regulation by GAPs and GEFs).
The specific roles of cAMP, kinases (PKA, PKC, JAK, Akt), phosphatases, calcium, calmodulin, PIP${2}$, IP${3}$, and DAG in signaling.
The detailed example pathways: ext{β2}-adrenergic receptor, Growth Hormone receptor, Epidermal Growth Factor receptor, and Insulin receptor.
How disruption or deregulation of signal transduction pathways contributes to various diseases, including bacterial infections and cancer.