Chapter 15 Part 1: Cell Signaling (Signal Transduction)
Mini lecture #1 covers Objectives 1-4
1. Understand the differences between contact dependent, paracrine, synaptic, and endocrine signaling. What is autocrine signaling?
Contact-Dependent Signaling
Requires direct membrane-to-membrane contact between cells.
The signaling molecule acts locally and is bound to signaling cells.
Paracrine Signaling
Signals act on neighboring cells and have limited range due to degradation by extracellular enzymes.
Can occur between different cell types or can refer to autocrine signaling when a cell responds to its own signals.
Autocrine Signaling
Cells send signals to themselves or to neighboring cells of the same type, resulting in coordinated responses.
Synaptic Signaling (Nerve Cells)
An action potential in an axon leads to neurotransmitter release at a synapse, transmitting the signal to a distant target cell.
Endocrine Signaling
Endocrine cells secrete hormones into the bloodstream, which transport hormones to distant target cells throughout the body.
2. Cells need combinations of signals to tell it what to do (grow and divide, differentiate). Be aware that most cells need to receive a variety of signals just to survive. What is apoptosis?
Signal Combinations
Cells are exposed to numerous signals, which activate specific combinations of receptors and pathways for appropriate responses.
The absence of necessary signals can lead cells to undergo apoptosis, a programmed form of cell death.
3. Be aware that different cells can respond differently to the same signal. For example, acetylcholine initiates different responses depending on the target cell. How is this possible?
Diverse Cellular Responses
Different cells can exhibit varied responses to the same ligand depending on receptor types and signaling machinery.
Example: Acetylcholine affects skeletal muscle contraction but decreases heart muscle contraction.
4. Extracellular signals bind to cell-surface receptors. Be familiar with the three major classes of cell surface receptors (ion channel linked, G protein linked, and enzyme linked).
Ion-Channel-Coupled Receptors: Transmitter (Ligand) Gated Ion Channels (e.g., Acetylcholine receptors at the neuromuscular junction).
G-Protein-Coupled Receptors (GPCRs): Act indirectly on membrane-bound target proteins via trimeric G-proteins. GPCRs are 7-pass transmembrane proteins.
Enzyme-Coupled Receptors: Function as enzymes or bind directly to enzymes, often activating protein kinases.
Mini lecture #2 covers Objectives 5-10
5. Cell surface receptors (G protein linked and enzyme linked) relay signals through a combination of small and large molecules. There is figure in your “notes” that illustrates how a signal is received by a cell surface receptor, transmitted across the cell, and relayed, amplified, integrated, spread and modulated till it reaches and alters an effector protein. You should be aware of the general ideas illustrated in this figure.
Intracellular Signaling Molecules:
Signals from cell-surface receptors are relayed into the interior through small and large signaling molecules.
Roles of Proteins in Signaling Pathways:
Proteins may:
Relay messages through the signaling chain.
Serve as scaffolds bringing together signaling proteins for interaction.
Transform or transduce the signal into different forms.
Amplify the received signal, increasing downstream responses.
Integrate signals from multiple pathways before relaying.
Spread signaling across different pathways.
Anchor signaling proteins at specific cellular sites.
Modify the activity of intracellular signaling proteins.
6. What is meant by the term “second messenger”? Give some examples.
Second Messengers
Small molecules generated in response to receptor activation.
Diffuse throughout the cell and alter the activity of selected effector proteins.
Examples include: cAMP (water-soluble), Ca$^{2+}$ (water-soluble), Inositol trisphosphate (IP3) (water-soluble), Diacylglycerol (lipid-soluble).
7. Understand how a cell can integrate more than one signal to activate a molecule in a signaling cascade.
Signal Integration
Complex cellular processes such as proliferation and survival necessitate the integration of multiple signals.
8. Intracellular signaling complexes form at activated receptors. How do scaffold proteins work to bring molecules into proximity?
Scaffold Proteins
Assist in assembling signaling complexes, either pre-formed or assembled post-signal recognition, thereby localizing signaling molecules to specific regions of the cell and enhancing interaction specificity.
9. What is meant by the statement: “receptor activation leads to modification of membrane phosphoinositides to create docking sites to assemble signaling complex interactions”?
Receptor activation can lead to the modification of membrane phosphoinositides, such as through phosphorylation, which creates specific docking sites. These sites are recognized by Modular Binding Domains like Plekstrin homology (PH) domains that bind to specific phosphorylated inositol phospholipids, thereby recruiting signaling proteins and assembling signaling complexes.
10. What is meant by the following protein domains (what do they bind to): SH2, SH3, PH, PTB? Understand how proteins can interact and be localized through these domains.
Modular Binding Domains
Facilitate signaling protein interactions and enhance specificity:
Plekstrin homology (PH) domains: Bind specific phosphorylated inositol phospholipids.
SRC Homology (SH2) domains: Bind to phosphorylated tyrosines.
SRC Homology 3 (SH3) domains: Bind to proline-rich peptide sequences.
(PTB domains are not explicitly mentioned in the provided notes.)
Mini lecture #3 covers Objectives 11-13
11. Some intracellular signaling proteins act as molecular switches. One class of molecular switches are protein phosphorylation. What enzymes are responsible for phosphorylating and dephosphorylating proteins? Which amino acids on signaling proteins are generally phosphorylated?
Phosphorylation-Based Switches
Activated by adding/removing phosphate groups.
Protein Kinases: Add phosphate groups (ATP-dependent).
Tyrosine Kinases (add to tyrosine residues).
Serine/Threonine Kinases (add to serine/threonine residues).
Protein Phosphatases: Remove phosphate groups (not ATP-dependent).
12. A second class of molecular switches are proteins that are GTP binding proteins. There are two types of GTP binding proteins, monomeric GTP binding proteins (recall, Ran, Sar1, Rab from Chapters 12 and 13) and, trimeric GTP-binding proteins? How are monomeric GTPases activated and inactivated?
GTP Binding Proteins
Second class ranging between GTP-bound (active) and GDP-bound (inactive) states.
Two types:
Small monomeric GTPases (e.g., Ran, Rab, Sar1).
Large trimeric G proteins (mediators of GPCR signaling).
Regulation of GTPases:
Guanine Nucleotide Exchange Factors (GEFs) facilitate GTP binding to reactivate GTP-binding proteins (activating).
GTPase-Activating Proteins (GAPs) promote GTP hydrolysis, switching GTPases to an an inactive state (inactivating).
13. What is meant by “desensitizing” a cell to a signal? What are some general ways a cell can become desensitized to a signaling molecule?
Adaptation and Desensitization
Cells can decrease their response to prolonged signals.
Mechanisms:
Receptor Sequestration: Internalizes receptors into recycling endosomes.
Receptor Down Regulation: Degrades receptors in lysosomes.
Receptor Inactivation: Keeps receptors inactivated in the plasma membrane.
Inactivation of Downstream Signaling Proteins: Downstream molecules may also be actively inactivated.
Production of Inhibitory Proteins: Prevents further signaling through pathway inhibition.
Mini lecture #4 covers Objectives 14-18
14. What is a G protein linked receptor (GPCR)? What is the general structure of the receptor (how many times does it cross the cell membrane)? GPCR’s are activated by extracellular signaling molecules (ligands). Be aware that there are many types of ligands that signal through GPCRs. Many different G-protein receptors can bind the same ligand (example; Adrenaline binds to nine different GPCRs).
G-Protein-Coupled Receptors (GPCRs)
The largest family of cell-surface receptors, mediating responses to external stimuli (sight, taste, etc.).
Consist of a single polypeptide chain that spans the membrane seven times (serpentine structure).
Activated by various extracellular signaling molecules (ligands).
15. BE AWARE THAT A G-PROTEIN IS NOT THE SAME THING AS A G PROTEIN COUPLED RECEPTOR (GPCR). What is a G protein (trimeric G protein). What is their general structure (what are the names of the 3 subunits that make up a G protein)? Which subunit is bound to GDP or GTP?
Trimeric G proteins
Act indirectly on membrane-bound target proteins upon activation by GPCRs.
Consist of three subunits: Gα, Gβ, and Gγ (implied by Gβγ dimer).
The Gα subunit is bound to either GDP (inactive state) or GTP (active state).
16. When do G-proteins interact with GPCRs? How are they activated upon ligand binding? What is the active form of G-proteins? What is the inactive form of G-proteins? Where are the three G-protein subunits found and how does that depend on GTP binding? How are G-proteins returned to their inactive form? What is meant by “intrinsic GTPase activity”? Understand that a regulator of G-protein signaling (RGS) acts as an an subunit-specific GTPase activating protein. Note the analogy to GAPS in “monomeric GTPases” we discussed earlier. After prolonged signaling, GPCRs can be “desensitized” (no longer responsive to signal).
G-Protein Activation and Inactivation
Interaction: Upon ligand binding, the activated GPCR interacts with trimeric G proteins on the cytoplasmic side of the membrane.
Activation: The activated GPCR causes the G protein to release GDP, allowing GTP to bind to the Gα subunit, thereby activating the G protein.
Active Form: The G protein dissociates into two active parts: the Gα subunit (with GTP) and the Gβγ dimer.
Inactive Form: The G protein is inactive when the Gα subunit is bound to GDP and is associated with the Gβγ dimer (trimeric form).
Subunit Location: In the inactive state, all three subunits (Gα-GDP, Gβ, Gγ) are associated with the membrane. Upon activation, the active Gα-GTP subunit and the active Gβγ dimer dissociate from each other and can engage downstream effectors.
Return to Inactive Form: The Gα subunit has intrinsic GTPase activity, meaning it hydrolyzes its bound GTP back to GDP. This causes the Gα-GDP subunit to reassociate with the Gβγ dimer, returning the G protein to its inactive trimeric state.
Intrinsic GTPase Activity: Refers to the Gα subunit's ability to hydrolyze GTP to GDP, which is a crucial step in turning off the G protein signal. (The note implies Gα's intrinsic activity for inactivation, without explicitly mentioning RGS proteins for trimeric G proteins, though the prompt makes an analogy to GAPs.)
17. What is the role of GPCR kinases (GRK) and arrestins in GPCR desensitization? How is arrestin recruited to the GPCR. What is the role of GPCR kinases (GRK’s) in this process. Understand that GRK’s can lead to endocytosis of GPCR’s by recruiting arrestin which can functin as an adaptin for endocytosis into a clathrin coated vesicle. After being endocytosed, a GPCR can either be recycled to the plasma membrane or degraded in a lysosome
GPCR Desensitization
After prolonged exposure to signaling, GPCRs can be phosphorylated by G-protein receptor kinases (GRKs).
This phosphorylation leads to the recruitment and arrestin binding to the GPCR, which inactivates the receptor and can lead to its potential internalization (endocytosis). The note states this can lead to internalization, but does not explicitly detail arrestin's role as an adaptin for clathrin-coated vesicles or the subsequent recycling/degradation pathways.
18. Be able to describe the general sequence involved in G Protein signaling (the video at the end of this section does a good job of this)
Ligand binding activates the GPCR, causing a conformational change.
Activated GPCR interacts with trimeric G proteins on the cytoplasmic side of the membrane.
G proteins release GDP, allowing GTP to bind, activating the G protein.
The G protein dissociates into two active parts: the Gα subunit (with GTP) and the Gβγ dimer.
These active components can then engage various downstream effectors (enzymes/ion channels).
Gα has GTPase activity, converting GTP back to GDP, thus resetting the G protein to its inactive state.
Mini lecture #5 covers Objectives 19-26
19. Cyclic AMP is an important second messenger. What is cyclic AMP (cAMP) and what is the reaction that leads to its formation? Which part of the cell do you expect cAMP to be found?
Cyclic AMP (cAMP)
An important water-soluble second messenger.
Formation: Produced via the enzymatic action of adenylyl cyclase, converting ATP into cAMP and pyrophosphate (PPi).
Location: As it is water-soluble, cAMP is found diffusing throughout the cytoplasm of the cell.
20. How is adenylyl cyclase activated by the G protein Gs? Where is adenylyl cyclase found? What reaction does it catalyze?
Adenylyl Cyclase
Activation: Stimulatory G proteins (Gs, a type of G protein) activate adenylyl cyclase, increasing intracellular cAMP.
Location: Adenylyl cyclase is typically a membrane-bound enzyme, where it can interact with membrane-associated G proteins.
Reaction: It catalyzes the conversion of ATP into cAMP and pyrophosphate.
21. What enzyme is responsible for removing cAMP from the cell and what is the chemical reaction it catalyzes?
Phosphodiesterases
These enzymes degrade cAMP to AMP, ceasing the signaling cascade and thereby removing cAMP from the cell.
22. Protein Kinase A (PKA) (also called “cyclic AMP-dependent protein kinase) is a serine/threonine kinase whose activation leads to different cellular responses. Be familiar with the steps that lead to activation of Protein Kinase A (PKA). What is the “second messenger” that activates protein kinase A (PKA) and what is the mechanism for this activation step in terms of regulatory and catalytic subunits of PKA? What enzyme reverses the effects of PKA?
Protein Kinase A (PKA)
A serine/threonine kinase.
Second Messenger: cAMP activates PKA.
Activation: The note states that cAMP activates PKA, leading to phosphorylation of target proteins. (The note does not explicitly detail the mechanism involving regulatory and catalytic subunits).
Reversing Enzyme: Phosphatases revert phosphorylated proteins (effects of PKA) to their inactive states.
23. Changes in gene expression are mediated by PKA. Where in the cell do you expect to find PKA in its inactive form? Upon activation where is PKA translocated to?
In its inactive form, PKA is typically found in the cytoplasm. Upon activation by cAMP, PKA can translocate to the nucleus to phosphorylate transcription factors, enabling changes in gene expression.
24. Understand the general roles of CRE, CREB and CREB binding protein (CBP) in gene transcription. What is a cyclic AMP response element (CRE)? Where is CRE found? A transcription regulator called CRE-binding (CREB) protein recognizes CRE elements. Once phosphorylated (by PKA), CREB recruits CREB binding protein (CBP). Understand the steps of this sequence of reactions. (the video at the end of this section does a good job of this)
cAMP-mediated Gene Expression
Cyclic AMP Response Element (CRE): A specific DNA sequence found in the regulatory regions of genes.
CRE-binding (CREB) protein: A transcription regulator that recognizes and binds to CRE elements.
Sequence of Reactions:
cAMP signaling activates PKA.
PKA translocates to the nucleus and phosphorylates CREB.
Phosphorylated CREB then recruits CREB binding protein (CBP).
The CREB-CBP complex leads to changes in gene transcription.
25. Signaling pathways produce transitory effects and hence must be “reset”. What is the role of protein phosphatases in turning off signaling pathways such as those initiated by cAMP?
Resetting the System
To turn off signaling pathways, protein phosphatases revert phosphorylated proteins to their inactive states, thereby maintaining cellular signaling balance and ceasing the effects initiated by pathways like those involving cAMP.
26. At this point in the course you should understand the functions (reactions catalyzed) by of each of the following classes of enzymes: phospholipases, kinases, phosphatases, GAP’s, GEF’s, GTPases, phosphatidyl inositol kinase (PIK), tyrosine kinase, serine/threonine kinase, protein phosphatases, phosphodiesterases and adenylyl cyclase.
Adenylyl cyclase: Converts ATP into cAMP and pyrophosphate (PPi).
Protein Kinases (general): Add phosphate groups to proteins (ATP-dependent).
Tyrosine Kinases: Add phosphate groups to tyrosine residues on proteins.
Serine/Threonine Kinases: Add phosphate groups to serine/threonine residues on proteins.
Protein Phosphatases: Remove phosphate groups from proteins (not ATP-dependent).
Phosphodiesterases: Degrade cAMP to AMP.
GTPase-Activating Proteins (GAPs): Promote GTP hydrolysis, switching GTPases to an inactive state.
Guanine Nucleotide Exchange Factors (GEFs): Facilitate GTP binding to reactivate GTP-binding proteins.
GTPases: Enzymes (like the Gα subunit's intrinsic activity) that hydrolyze GTP to GDP.
(Phospholipases and phosphatidyl inositol kinase (PIK) are not explicitly defined in terms of their catalyzed reactions within the provided notes.)