Unicellular Organisms

● Early unicellular organisms developed responses to environmental changes, including

reactions to other cells.
● Present-day unicellular organisms, like bacteria and yeasts, communicate and influence
each other's behavior.
● Bacteria use "quorum sensing" for coordinated actions based on chemical signals from
neighboring bacteria, especially at higher population densities.
● Yeast cells, like Saccharomyces cerevisiae, release mating factors to prepare for mating
and produce a diploid zygote upon fusion.

Evolution of Intercellular Communication:

● Multicellular organisms possess sophisticated intercellular communication systems.
● Cell well-being is often subordinated to the benefit of the entire organism in these tight-
knit cell communities.
● Intercellular communication enables diverse tissues and cell types to collaborate and
coordinate functions during development and adulthood.

Communication in Multicellular Organisms:

● Extracellular signal molecules mediate communication between cells in multicellular
organisms.
● Some signals act over long distances to reach cells far away, while others signal only
● to immediate neighbors.
● Most cells in multicellular organisms both emit and receive signals.
● Signal reception depends on receptor proteins, usually found at the cell surface,
which bind the signal molecule, leading to receptor activation.
● The activated receptor then triggers one or more intracellular signaling pathways.
● Intracellular signaling proteins process the incoming signal and distribute it to the
appropriate intracellular targets, known as effector proteins.
● Effector proteins, modified by the incoming signal, implement the necessary changes
● in cell behavior.
● These effectors can be various components, including transcription regulators, ion
channels, metabolic pathway constituents, or cytoskeleton elements.

Conservation of Cell Signaling:

● Cell signaling features are conserved across eukaryotic evolution.
● Unicellular organisms like budding yeast utilize cell-surface receptors, intracellular GTP-
binding proteins, and protein kinases in responding to mating factors.
● Animal signaling systems have evolved to be considerably complex through gene
duplication, divergence, and diversification.
● This complexity is reflected in a diverse array of receptor proteins, further enhanced by
processes like alternative RNA splicing and post-translational modifications.
● Human genome contains over 1500 genes encoding receptor proteins, underscoring the
complexity of these signaling systems.
 Types of signal transmission :
● Multicellular organisms use four categories of chemical signaling: paracrine, endocrine,
autocrine, and direct signaling via gap junctions.
● Paracrine signaling involves local signals between nearby cells, moving via diffusion
through the extracellular matrix and generating quick, short-lived responses.

Synaptic Signaling
● Synaptic signaling, a form of paracrine signaling, occurs at chemical synapses between
nerve cells, transmitting signals through neurotransmitters released at the synapse. The
electrochemical potential of the target cell changes upon neurotransmitter binding to
receptors, initiating the next electrical impulse. Neurotransmitters are swiftly degraded or
reabsorbed after release.

Extracellular signals can act over short or long distances:

- Extracellular signals can act over short or long distances.
- Extracellular signal molecules may stay bound to the signaling cell's surface, affecting only
nearby cells.
- Contact-dependent signaling is crucial in development and immune responses, where cells
extend processes to make contact, sometimes over substantial distances.

Four forms of intercellular signaling:

a) Contact-dependent signaling requires direct membrane-to-membrane cell contact.
b) Paracrine signaling involves local mediators released into the extracellular space, affecting
nearby cells.
c) Synaptic signaling uses neurotransmitters at synapses, often distant from the neuronal cell
body.
d) Endocrine signaling involves endocrine cells secreting hormones into the bloodstream to
reach distant target cells.
Common signaling molecules used in paracrine, synaptic, and endocrine signaling, differing in
speed and selectivity.
Paracrine signaling involves local mediators acting on nearby cells, with the signaling and target
cells being of different types.
Autocrine signaling occurs when cells produce signals that affect themselves.
Cancer cells often produce signals stimulating their own survival and proliferation.
Organisms have specialized cell types, such as neurons and endocrine cells, for long-range
intercellular communication.
Neurons use electrical impulses (action potentials) and neurotransmitters at synapses to
transmit signals over long distances.
Endocrine cells secrete hormones into the bloodstream for widespread distribution and distant
target cell interaction.
Extracellular Signal Molecules Bind to Specific Receptors:
Extracellular signal molecules facilitate communication between cells in multicellular animals.
Signal molecules range from proteins, peptides, and amino acids to gases like nitric oxide and
carbon monoxide.
Release of signal molecules occurs through exocytosis, diffusion through plasma membrane, or
display on cell surfaces.
Four forms of intercellular signaling: contact-dependent, paracrine, synaptic, and endocrine,
differing in speed and selectivity.
Target cells respond to signals via specific receptors with high specificity to recognize
appropriate signals.
Receptors, often transmembrane proteins, activate and generate intracellular signals upon
binding extracellular signal molecules (ligands).
Receptor location varies, with some on the target-cell surface and others inside, necessitating
ligand diffusion across the plasma membrane.

Each Cell Is Programmed to Respond to Specific Combinations of

Extracellular Signals:
- Cells in multicellular organisms are exposed to a variety of signal molecules in their
environment, which can be in soluble form, bound to the extracellular matrix, or attached to
neighboring cell surfaces.
- Cells selectively respond to specific signals by expressing receptors and intracellular signaling
systems tailored to the necessary regulation.
- Cells respond to multiple signals in their environment, and interactions between these signals
influence cellular responses crucial for decisions like division, movement, and differentiation.
- Many cells require specific combinations of extracellular survival factors to continue living, and
deprivation of these essential signals can activate programmed cell death (apoptosis).
- Animals use countless combinations of different signal molecules to control diverse cell
behaviors in highly specific ways, reflecting the complexity of how cells respond to signal
combinations.
- A single signal molecule can have different effects on various types of target cells due to
variations in intracellular signaling proteins, effector proteins, and activated genes, reflecting the
importance of the cell's predetermined state and developmental history.

Three Major Classes of Cell-Surface Receptor Proteins:

- Extracellular signal molecules bind to specific cell-surface receptors on target cells, triggering
intracellular signal modification.
- Three classes of cell-surface receptor proteins:
1. Ion-Channel-Coupled Receptors: Involved in rapid synaptic signaling, altering ion
permeability briefly.
2. G-Protein-Coupled Receptors: Indirectly regulate plasma-membrane-bound target proteins
via trimeric GTP-binding proteins, influencing intracellular signaling molecules and ion
permeability.
3. Enzyme-Coupled Receptors: Function as enzymes or associate with enzymes, activating
signaling cascades by phosphorylating specific proteins.
- Specialized receptors that play crucial roles in cell specialization, tissue renewal, and repair do
not fit neatly into the three mentioned classes. Further discussion follows the explanation of G-
protein-coupled receptors and enzyme-coupled receptors.

Cell-Surface Receptors Relay Signals Via Intracellular Signaling Molecules:

- Intracellular signaling molecules transmit signals from cell-surface receptors, modulating
effector proteins and cell behavior.
- Second messengers, like cyclic AMP and Ca2+, act as intermediaries between extracellular
and intracellular components.
- Protein signaling molecules, often acting as molecular switches, activate downstream proteins
or generate second messengers.
- Phosphorylation is a critical process, where protein kinases activate switches by adding
phosphate groups, while phosphatases deactivate them.
- Protein kinases can be serine/threonine or tyrosine kinases, organizing into kinase cascades
to transmit or amplify signals.
- GTP-binding proteins, including trimeric G proteins and monomeric GTPases, serve as
molecular switches, transitioning between active and inactive states.
- Regulatory proteins like GTPase-activating proteins (GAPs) and guanine nucleotide exchange
factors (GEFs) control GTP-binding protein states.
- Other switches can be activated or deactivated by various factors, including covalent
modifications and signaling proteins.
- Signaling pathways may involve inhibitory steps, contributing to the complexity of the system.
Double-negative activation is a common feature in signaling systems.

Intracellular signals must be specific and precise in a noisy cytoplasm:

- Intracellular signaling aims for specificity despite a crowded cytoplasm prone to cross-talk and
interference.
- Mechanisms for signal specificity rely on high affinity, specific interactions, and docking sites
between signaling molecules and correct partners.
- Unwanted signals are filtered out through downstream target proteins, responding only to
specific concentration or activity levels of upstream signals.
- Constitutively active protein phosphatases minimize the impact of background noise by
removing unwanted phosphorylation.
- Signaling systems are designed for robust and precise responses, utilizing backup
mechanisms such as parallel pathways and redundancy.
- Redundancy ensures response even if one signaling pathway is compromised, contributing to
adaptability and resilience in intracellular signaling.

Intracellular Signaling Complexes Form at Activated Receptors:

- Localizing signaling molecules within the same cellular compartment or large protein
complexes enhances interaction specificity.
- Scaffold proteins play a crucial role in localizing and bringing signaling proteins into close
proximity, enabling rapid and selective activation in response to signals.
- Scaffold proteins help prevent unwanted cross-talk with other signaling pathways, ensuring
correct signals reach appropriate downstream targets.
- Signaling complexes can form transiently in response to extracellular signals and disassemble
when the signal is no longer present.
- Receptor activation often involves phosphorylation of the receptor's cytoplasmic tail, creating
docking sites for assembly of other signaling proteins.
- Phosphoinositides, modified phospholipid molecules produced upon receptor activation, act as
recruiting signals, attracting specific signaling proteins to specific membrane regions and
initiating intracellular signaling cascades.
- The formation of intracellular signaling complexes, facilitated by scaffold proteins and involving
receptor phosphorylation and phosphoinositides, enhances specificity and efficiency in cellular
signal transduction.

Modular interaction domains mediate interactions between intracellular signalling proteins:

- Induced proximity activates intracellular signaling proteins by bringing them close together.
- Signaling complexes rely on interaction domains, small conserved domains binding specific
motifs in other proteins or lipids.
- Interaction domains include SH2, PTB, SH3, and PH domains, enabling versatile binding and
pathway formation.
- Concentrating receptors and signaling proteins in specific cell regions, like primary cilia, also
facilitates signaling.
- Characteristics of intracellular signaling systems vary in response timing, sensitivity, dynamic
range, persistence, signal processing, integration, and coordination of multiple responses.
- Signaling systems form complex network structures, rarely linear, with information flow in
multiple directions, presenting a significant research challenge.

The speed of a response depends on the turnover of signalling

Molecules:
- Speed of signaling response is influenced by the nature of intracellular signaling molecules.
- Rapid responses occur with pre-existing proteins, such as allosteric changes in ion channels
altering membrane potential in milliseconds.
- Protein phosphorylation-based responses occur within seconds.
- Gene expression changes and new protein synthesis lead to responses taking minutes to
hours.
- Transient extracellular signals can have lasting effects in development through cell memory
mechanisms.
- In most adult tissues, responses fade when signals cease.
- Turnover rate of intracellular molecules affected by the signal determines response
promptness.
- Response speed differs based on the turnover rates of signaling molecules.
- Inactivation processes, like phosphorylation or GTP hydrolysis, play a crucial role in signaling
response speed, magnitude, and duration.
Cells Can Respond Abruptly to a Gradually Increasing Signal:
- Signaling systems exhibit different response types based on signal concentration.
- Smoothly graded responses fine-tune metabolic processes with hormones.
- Some systems respond significantly only when the signal surpasses a threshold.
- Abrupt responses can be sigmoidal (steep response) or discontinuous (all-or-none).
- Discontinuous responses are useful for binary cell state decisions and involve positive
feedback.
- Sigmoidal responses involve multiple signaling molecules binding downstream to induce a
response.
- Sharpening of response occurs with increased molecules or phosphate groups.
- Response sharpening involves activation of one enzyme and inhibition of another with
opposing functions.
- Example: Adrenaline binding to G-protein-coupled receptors increases cyclic AMP, activating
glycogen breakdown and inhibiting glycogen synthesis.

Positive feedback can generate an All-or-none response:

- Intracellular signaling systems commonly use feedback loops, resembling metabolic and gene
control pathways.
- Feedback loops can be either positive (output stimulates its own production) or negative
(output inhibits its own production).
- Positive feedback intensifies the response to a signal, often resulting in sigmoidal responses.
- Strong positive feedback can lead to an all-or-none response and bistability, sustaining the
response even with a weakened signal.
- Positive feedback enables transient signals to induce long-term changes in cells without
altering DNA.
- Muscle-cell specification involves positive feedback loops activating muscle-specific genes and
transcription regulatory proteins.
- Positive feedback in this process creates a permanent change in cell identity without altering
DNA sequences.
- Studies focusing on cell populations may mask abrupt, discontinuous responses seen in single
cells due to intrinsic variability in signaling systems, especially at intermediate signal
concentrations.
- All-or-none responses are evident at the single-cell level, highlighting the individual cell's
intrinsic behavior in signaling.

Negative feedback is a common motif in signalling systems:

- Negative feedback opposes and limits the response to a stimulus, reducing system sensitivity.
- Delayed negative feedback can induce oscillatory responses, which may continue even
without external stimuli.
- Many oscillators with oscillatory behavior feature positive feedback loops.
- Short-delay negative feedback results in adaptation, characterized by a strong initial response
to a stimulus followed by rapid decay.
- Adaptation functions as a change detector, responding strongly to sudden increases in
stimulus.

Cells can adjust their sensitivity to a signal:

- Adaptation allows cells to respond to changes in signal molecule concentration rather than
absolute concentration.
- Mechanisms of adaptation involve inactivation or sequestration of receptors, receptor down-
regulation, and intracellular signaling modifications.
- Intracellular signaling systems are complex, involving multiple cross-regulatory pathways and
feedback loops.
- The signaling network serves as a computing device for the cell, processing a wide range of
signals.
- Understanding the system's behavior as a whole is challenging due to the quantitative details
of molecular interactions.
- Future research aims to develop advanced quantitative and computational methods for in-
depth system analysis.

Summary:
Each cell in a multicellular organism responds to specific extracellular signal molecules through
complementary receptors. These receptors, often cell-surface proteins, transduce external
signals into intracellular responses, modifying the target cell's behavior. Activated receptors
initiate intracellular signaling cascades, involving diverse signaling proteins that transduce,
amplify, integrate, and relay the signals. Some act as transient switches activated by
phosphorylation or GTP binding. Signaling proteins form large complexes using modular
interaction domains, enabling the formation of functional signaling networks. These mechanisms
help cells interpret and respond to a variety of signaling inputs, including feedback mechanisms
to regulate responses.

**Target Cell Response Mechanisms:**

- **Positive Feedback Loops:**

- Enable all-or-none responses to increasing signal concentrations.
- Convert short-lasting signals into long-lasting or irreversible responses.

- **Negative Feedback:**
- Allows cells to adapt to signal molecule changes, responding to a wide concentration range.
- Shortens and limits the response level, reducing sensitivity to perturbations.

**Signal Transmission through G Protein-Coupled Receptors:**

- **GPCRs Overview:**
- Over 800 GPCRs in humans, involved in various sensory perceptions and responses.
- Respond to diverse ligands, including proteins, peptides, photons, and more.
- Activate multiple GPCR family members, leading to different responses.
- **G Protein Signaling:**
- GPCRs activate G proteins (α, β, γ subunits) that relay signals to enzymes or ion channels.
- GTP-GDP exchange on α subunit triggers signaling cascade, with subsequent target
activation.
- RGS proteins act as GTPase-activating proteins, aiding in G protein signaling regulation.

- **cAMP Signaling:**
- cAMP, a second messenger, modulates cellular responses via PKA activation.
- Synthesized from ATP by adenylyl cyclase, regulated by G proteins.
- Gs and Gi proteins modulate cAMP levels by activating or inhibiting adenylyl cyclase.

**Calcium as a Ubiquitous Intracellular Mediator:**

- **Ca2+ Functions:**
- Induces varied cellular responses upon signal activation.
- Essential for processes like muscle contraction and synaptic transmission.

- **Ca2+ Waves and Oscillations:**

- Ca2+-induced Ca2+ release amplifies Ca2+ signals through positive feedback.
- Oscillations occur due to negative feedback and influence cellular responses.

- **CaM-Dependent Kinases:**
- Calmodulin (CaM) activates Ca2+/CaM-dependent protein kinases.
- Phosphorylate target proteins in response to Ca2+ signals, affecting various cellular
functions.

**Nitric Oxide (NO) Signaling:**

- **NO as a Signaling Molecule:**

- Diffusible gas molecule that acts as a signaling mediator between cells.
- Induces relaxation of smooth muscle cells, impacting blood vessel dilation and heart rate.

- **NO Synthesis and Functions:**

- Produced by nitric oxide synthases (NOS) through arginine deamination.
- Stimulates cyclic GMP synthesis, influencing various physiological responses.

**Amplification of Signals and Desensitization:**

- **Signal Amplification:**
- Signaling cascades involving relay chains and second messengers amplify responses to
extracellular signals.
- Enables a single extracellular signal molecule to impact numerous intracellular proteins.
- **GPCR Desensitization:**
- Involves receptor phosphorylation and arrestin binding upon prolonged ligand exposure.
- Modifies receptor molecules, preventing interactions with G proteins and promoting
internalization or inactivation.

SIGNALLING THROUGH ENZYME-COUPLED RECEPTORS:

**Enzyme-Coupled Receptors:**

- Transmembrane proteins with ligand-binding domain on plasma membrane outer surface.

- Lack association with trimeric G proteins; possess intrinsic or associated enzyme activity in
cytosolic domain.
- Typically have one transmembrane segment per subunit, contrasting with GPCRs (seven
segments).
- Activate similar signaling pathways as GPCRs.
- Focus on Receptor Tyrosine Kinases (RTKs), the most common type of enzyme-coupled
receptors.

**Receptor Tyrosine Kinases (RTKs):**

- Many extracellular signal proteins act through RTKs.

- Control cell behavior in both developing and adult animals.
- Approx. 60 human RTKs categorized into 20 structural subfamilies, each specific to certain
protein ligands.

**Activation of RTKs:**

- Ligand binding to extracellular domain activates cytosolic tyrosine kinase domain.

- Activation leads to phosphorylation of tyrosine side chains on the cytosolic part.
- Phosphorylated tyrosines create docking sites for intracellular signaling proteins, relaying the
signal.

**Activation Mechanism:**

- Unlike GPCRs, RTKs mainly dimerize upon ligand binding.

- Dimerization brings cytoplasmic kinase domains in close proximity, promoting activation.
- Dimerization-induced activation stimulates various mechanisms depending on the RTK type.
- e.g., Insulin receptor dimerization enables phosphorylation of specific tyrosines, inducing
conformational changes and full activation.
- e.g., Epidermal Growth Factor (EGF) receptor kinase is activated by conformational changes
resulting from interactions between kinase domains outside their active sites.

Phosphorylated Tyrosines on RTKs Serve as Docking Sites for Intracellular Signaling Proteins:
**RTK Activation and Phosphorylation:**
- Upon activation, RTK kinase domains phosphorylate multiple cytosolic receptor sites.
- Phosphorylation occurs in disordered regions outside the kinase domain.
- Creates high-affinity docking sites for intracellular signaling proteins.
- Specific phosphotyrosine-binding domains in signaling proteins bind to phosphorylated sites,
triggering activation.
- Binding to activated RTK can phosphorylate and activate signaling proteins.
- Binding alone can induce conformational changes or proximity to the next protein in the
signaling pathway.

**Assembly of Intracellular Signaling Complex:**

- Receptor phosphorylation triggers assembly of intracellular signaling complex.

- Complex relays the signal along various routes within the cell.

**Diverse Responses from Different RTKs:**

- Different RTKs bind distinct signaling protein combinations, leading to varied cellular
responses.

**Additional Docking Proteins:**

- Some RTKs utilize additional docking proteins to enhance signaling complex size and
complexity.
- Insulin and IGF1 receptor signaling rely on specialized docking protein IRS1.
- IRS1 associates with phosphorylated tyrosines on activated receptor, getting phosphorylated
at multiple sites, creating more docking sites.

Proteins with SH2 domains bind to phosphorylated tyronises

**Binding of Intracellular Signaling Proteins:**

- Intracellular signaling proteins bind phosphotyrosines on activated RTKs or docking proteins

like IRS1.
- Signal relay through protein-protein interactions via modular interaction domains.
- Enzymes such as PLCγ, cytoplasmic tyrosine kinase Src, and PI 3-kinase dock on these
receptors.
- PLCγ activates inositol phospholipid signaling, elevating cytosolic Ca2+ and activating PKC.
- PI 3-kinase phosphorylates lipids, creating docking sites for other signaling proteins at the
plasma membrane.

**Phosphotyrosine-Binding Domains:**

- Signaling proteins contain phosphotyrosine-binding domains (e.g., SH2, PTB) to bind activated
RTKs and phosphorylated intracellular signaling proteins.
- These domains facilitate binding to tyrosine-phosphorylated proteins transiently.
**Negative Feedback Mechanisms:**

- Proteins binding activated RTKs via SH2 domains contribute to negative feedback, reducing
signaling.
- Example: c-Cbl catalyzes receptor ubiquitylation, promoting endocytosis and degradation
(receptor down-regulation).

**Endocytosis and Signaling:**

- Endocytosis of RTKs, similar to GPCRs, doesn't always decrease signaling; RTKs can signal
from endosomes or other intracellular compartments.
- Example: NGF binds TrkA RTK, and signaling endocytic vesicles transport signals to the cell
body.

**Adaptor Proteins:**

- Some signaling proteins, rich in SH2 and SH3 domains, act as adaptors, linking tyrosine-
phosphorylated proteins to those lacking SH2 domains.
- Adaptor proteins facilitate coupling of activated RTKs to important signaling protein Ras,
activating downstream pathways.

The GTPase Ras Mediates signaling by Most RTKs:

**Ras Superfamily:**

- Ras superfamily includes distinct families of monomeric GTPases, with Ras and Rho families
being key in relaying signals from cell-surface receptors.
- Ras and Rho GTPases act as central signaling hubs, spreading signals across multiple
downstream pathways.

**Ras Proteins in Humans:**

- Three major Ras proteins in humans: H-Ras, K-Ras, N-Ras.

- Subtle functional differences, collectively referred to as Ras.
- Ras contains lipid groups anchoring it to the membrane, facilitating signal relay.
- Crucial for RTK-mediated signaling to the nucleus, stimulating cell proliferation and
differentiation.
- Hyperactive mutant Ras in 30% of human tumors leads to uncontrolled cancer cell
proliferation.

**Molecular Switch Function:**

- Ras functions as a molecular switch, transitioning between active (GTP-bound) and inactive
(GDP-bound) states.
- Activity regulated by signaling proteins influencing this transition.

**Ras Regulation:**

- Ras-GEFs stimulate GDP dissociation and GTP uptake, activating Ras.

- Ras-GAPs enhance GTP hydrolysis, inactivating Ras.
- Hyperactive Ras mutants resist GAP-mediated GTPase stimulation, contributing to cancer.

**RTK Activation of Ras:**

- RTKs activate Ras via Ras-GEF or inhibiting Ras-GAP.

- Ras-GEFs indirectly coupled to RTKs, driving Ras into an active state.
- Loss of Ras-GEF function mimics Ras loss.
- Ras superfamily activation involves GEFs and specific downstream protein activation.

**Discovery of Ras Activation Mechanism:**

- RTKs activate Ras via genetic studies in Drosophila (discovery through Sev and Sos genes).
- Grb2 and Sos link activated RTKs to Ras in mammalian cells, indicating a highly conserved
mechanism.

**Ras Activation and Signal Relay:**

- Activated Ras triggers various signaling proteins to relay the signal downstream.

Ras Activates a MAP Kinase Signaling Module:

**Short-Lived Tyrosine Phosphorylations and Ras Activation:**

- Activated RTKs trigger short-lived tyrosine phosphorylations and Ras activation.

- Tyrosine-specific protein phosphatases rapidly reverse tyrosine phosphorylations.
- Ras-GAPs induce inactivation of activated Ras.
- Result in graded or switchlike cellular responses, varying in duration.

**MAP Kinase Positive Feedback and Negative Feedback:**

- MAP kinase can activate positive feedback loops, influencing irreversible processes.
- MAP kinases activate negative feedback loops by increasing phosphatase concentration.
- Negative feedback loops involve phosphatase gene transcription and enzyme stabilization.
- Erk phosphorylates and inactivates Raf, creating a negative feedback loop in the Ras-MAP-
kinase pathway.

**Scaffold Proteins and MAP Kinase Signaling Modules:**

- Three-component MAP kinase signaling modules are prevalent in eukaryotic cells.

- Different modules in the same cell mediate various responses.
- Scaffold proteins prevent cross-talk between parallel MAP kinase modules.
- Scaffold strategy ensures response specificity but limits signal amplification and dispersion.

**MAP Kinase Signaling Modules in Mammalian Cells:**

- Mammalian cells employ scaffold proteins to prevent cross-talk in MAP kinase modules.
- There are at least five parallel MAP kinase modules in mammalian cells.
- Modules respond to various stresses or signals from neighboring cells.
- Trade-off between precision and signal amplification due to the scaffold strategy.

These notes summarize essential information regarding short-lived tyrosine phosphorylations,

Ras activation, feedback mechanisms in MAP kinase signaling, the role of scaffold proteins, and
the trade-off involved in ensuring response specificity in MAP kinase signaling.

RHO Family GTpases functionally couple cell-surface receptors to the cytoskeleton:

**Rho Family GTPases:**

- Rho family GTPases belong to the Ras superfamily and relay signals from cell-surface
receptors.
- They regulate actin and microtubule cytoskeletons, impacting cell shape, polarity, motility,
adhesion, cell-cycle progression, gene transcription, and membrane transport.
- Key members: Rho, Rac, and Cdc42, affecting multiple downstream target proteins.
- Activated by GEFs and inactivated by GAPs, with numerous GEFs and GAPs in humans.

**Inactive Rho Family GTPases:**

- Inactive Rho family GTPases are often bound to guanine nucleotide dissociation inhibitors
(GDIs) in the cytosol, preventing interaction with Rho-GEFs at the plasma membrane.

**Ephrin Signaling Example:**

- Ephrin signaling exemplifies how RTKs can activate a Rho GTPase.

- Ephrins activate Eph family RTKs, influencing motor neuron axon guidance.
- Eph receptor activation leads to growth cone collapse, guided axon extension.
- Involves Rho-GEF ephexin associated with Eph receptor's cytosolic tail.
- Eph receptor activation enhances ephexin's ability to activate RhoA, impacting actin
cytoskeleton and growth cone collapse.

These notes provide a concise overview of Rho family GTPases, their activation and
inactivation mechanisms, and a specific example of Rho GTPase activation in Ephrin signaling.
PI 3-Kinase Produces Lipid Docking Sites in the Plasma Membrane:
**Phosphoinositide 3-Kinase (PI 3-Kinase):**

- Plasma-membrane-bound enzyme associated with RTK intracellular tails.

- Phosphorylates inositol phospholipids, activated by RTKs and GPCRs.
- Central role in promoting cell survival and growth.

**Phosphoinositides and PI 3-Kinase:**

- Phosphatidylinositol undergoes reversible phosphorylation to generate phosphoinositides.

- PI 3-kinase phosphorylates at the 3 position, forming phosphoinositides, with PI(3,4,5)P3
being significant.
- PI(3,4,5)P3 serves as a docking site for intracellular signaling proteins.

**Contrast with Previous Phosphoinositide Use:**

- Unlike PI(4,5)P2 in GPCR and RTK signaling, PI(3,4,5)P3 is not cleaved by phospholipases.
- PI(3,4,5)P3 remains until specific phosphatases dephosphorylate it.
- PTEN phosphatase dephosphorylates PI(3,4,5)P3; mutations linked to uncontrolled cell
growth.

**Types of PI 3-Kinases:**

- Class I PI 3-kinases activated by RTKs and GPCRs.

- Class Ia activated by RTKs, regulatory subunit is an adaptor protein.
- Class Ib activated by GPCRs, regulatory subunit binds to βγ complex of activated G protein.
- Common catalytic subunit can also be activated by direct Ras binding.

**Binding of Signaling Proteins to PI(3,4,5)P3:**

- Intracellular signaling proteins bind to PI(3,4,5)P3.

- Use specific interaction domains like pleckstrin homology (PH) domains.
- PH domains primarily for protein–protein interactions, some also recognize specific
membrane-bound proteins.

**PH Domains in Human Proteins:**

- Found in about 200 human proteins, including Ras-GEF Sos.

**Role of Akt (Protein Kinase B):**

- Akt is a serine/threonine protein kinase with a PH domain.

- PI-3-kinase–Akt pathway activated by insulin, promoting cell survival and growth.
These notes summarize the role of PI 3-kinase, phosphoinositide phosphorylation, PI(3,4,5)P3
importance, contrast with previous phosphoinositides, types of PI 3-kinases, binding of signaling
proteins to PI(3,4,5)P3, PH domains, and Akt in the PI-3-kinase–Akt signaling pathway.

RTKs and GPCRs activate overlapping signaling pathways:

**Shared Signaling Pathways between RTKs and GPCRs:**

- RTKs and GPCRs activate common intracellular signaling pathways.

- Both receptor types can activate the inositol phospholipid pathway mediated by phospholipase
C.

**Convergence of Different Pathways onto Common Target Proteins:**

- Even when RTKs and GPCRs activate distinct pathways, these pathways can converge on the
same target proteins.

**Interactions among Parallel Signaling Pathways:**

- Pathways triggered by GPCRs, RTKs, or both types of receptors.

- Interactions and crosstalk among these pathways allow different extracellular signals to
modulate and coordinate their effects.

These notes highlight the presence of shared signaling pathways between RTKs and GPCRs
and how different pathways, when activated by these receptors, can converge on common
target proteins. It emphasizes the importance of interactions among parallel signaling pathways
to modulate and coordinate cellular responses to various extracellular signals.

Some enzyme -couples receptors associate with cytoplasmic tyronose kinses:

**Tyrosine Phosphorylation-Dependent Cell-Surface Receptors:**

- Receptors dependent on tyrosine phosphorylation for activity.

- Lack a tyrosine kinase domain and use cytoplasmic tyrosine kinases for phosphorylation.
- Function similarly to RTKs with a separate gene-encoded kinase domain.

**Cytoplasmic Tyrosine Kinases:**

- Many dependent on Src family, a major group of mammalian cytoplasmic tyrosine kinases.
- Src family includes Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk with SH2 and SH3 domains.
- Located on the cytoplasmic side, interacting with transmembrane receptors and lipid chains.
- Activate upon ligand binding, phosphorylating distinct target proteins.

**Activation of Cytoplasmic Tyrosine Kinases:**

- Activated by extracellular ligand binding to appropriate receptor proteins.

**Integration of Signaling Pathways:**

- Receptors like integrins can cluster and activate intracellular signaling pathways.
- Focal adhesion kinase (FAK) plays a role by binding to integrins and interacting with Src
kinases.

This paragraph outlines receptors relying on tyrosine phosphorylation, utilizing cytoplasmic

tyrosine kinases like the Src family. It emphasizes their role in signaling pathways and
integration, notably in focal adhesions with integrins.

Cytoskine receptors activate the JAK-STAT signalling pathway:

**Cytokine Receptors and JAK-STAT Signaling Pathway:**

- Cytokine receptors for cytokines and certain hormones.

- Associated with cytoplasmic tyrosine kinases (JAKs) and STAT transcription regulators.
- JAK-STAT pathway: direct route from cell-surface receptors to nucleus for gene transcription.

**Cytokine Receptors and JAKs:**

- Cytokine receptors form dimers or trimers, associating with JAKs (JAK1, JAK2, JAK3, Tyk2).
- Cytokine binding leads to JAK mutual phosphorylation and increased tyrosine kinase activity.
- JAKs phosphorylate cytokine receptor cytoplasmic tails, creating phosphotyrosine docking
sites for STATs.

**STAT Proteins and Their Functions:**

- At least six mammalian STATs, each with an SH2 domain.

- SH2 domain: binds phosphotyrosine docking sites on activated cytokine receptors, mediates
STAT interactions.
- JAK-phosphorylated STATs form homodimers or heterodimers, translocate to the nucleus, and
stimulate transcription.

**Examples of Cytokines and Hormones Activating JAK-STAT Pathway:**

- Over 30 cytokines and hormones activate JAK-STAT pathway by binding to cytokine

receptors.

**Negative Feedback Mechanisms:**

- JAK-STAT pathway regulated by negative feedback.

- STAT dimers activate genes encoding inhibitory proteins, terminating the response.
- Inhibitory proteins bind to phosphorylated JAKs, receptors, or STAT dimers to prevent DNA
binding.
- Dephosphorylation of phosphotyrosines on activated JAKs and STATs required to turn off the
response.

Protein tyrosine Phosphatases reverse tyrosine phosphorykations:

**Tyrosine Phosphorylation and Protein Tyrosine Phosphatases:**

- Protein tyrosine phosphatases reverse tyrosine phosphorylation in signaling pathways.

- They are equally important as kinases in signaling.
- About 100 protein tyrosine phosphatases encoded in the human genome.
- Some are dual-specificity phosphatases, dephosphorylating serines and threonines as well.
- Exist in both cytoplasmic and transmembrane forms.
- Most exhibit selective specificity, removing phosphates from specific phosphotyrosines.
- Regulated to act at appropriate time and location, maintaining low levels of tyrosine
phosphorylation.

**Receptor Serine/Threonine Kinases and Smads:**

- Some receptors use serine and threonine phosphorylation instead of tyrosine phosphorylation.
- Activate a direct signaling pathway to the nucleus involving phosphorylation of Smads.
- Phosphorylated Smads translocate to the nucleus, regulating gene transcription.

Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and
Smads:
Here are the bullet points summarizing the provided paragraphs:

**The TGFβ Superfamily:**

- Comprises 33 structurally related, secreted, dimeric proteins.

- Regulate various biological functions, including pattern formation, cell behaviors, extracellular
matrix production, and cell death.
- Divided into TGFβ/activin family and BMP family.

**Receptor Serine/Threonine Kinases:**

- Work through enzyme-coupled receptors with serine/threonine kinase domains.

- Two classes: type I and type II receptors, structurally similar homodimers.
- Activate by binding specific combinations of type-I and type-II receptor dimers.

**Signal Relay to the Nucleus:**

- Activated type-I receptors phosphorylate Smad transcription regulators.

- Phosphorylated receptor-activated Smads (R-Smads) dissociate from the receptor and bind to
Smad4 to form a complex that translocates into the nucleus.
- Smad complex associates with other transcription regulators to control gene transcription.

**Endocytosis and Inactivation:**

- Activated TGFβ receptors and ligands endocytosed through two routes: activation and
inactivation.
- Activation route depends on clathrin-coated vesicles, leading to early endosomes where Smad
activation occurs.
- Inactivation route depends on caveolae, leading to receptor ubiquitylation and degradation.

**Continuous Shutting Between Cytoplasm and Nucleus:**

- Smads shuttle between cytoplasm and nucleus, dephosphorylated in the nucleus and exported
to the cytoplasm for rephosphorylation.
- Gene response depends on extracellular signal concentration and signal exposure duration.

**Negative Feedback:**

- Target genes encode inhibitory Smads, Smad6, or Smad7, providing negative feedback in the
Smad pathway.

**Cross-talk with Other Signaling Pathways:**

- Receptor serine/threonine kinases can stimulate other intracellular signaling proteins like MAP
kinases and PI 3-kinase.
- Proteins from other signaling pathways can phosphorylate Smads and influence the Smad
pathway.

Here are the concise bullet points for each section:

**Alternative Signaling Routes in Gene Regulation:**

*Introduction:*
- Cells' behaviors influenced by gene expression changes.
- Extracellular signaling molecules affect gene expression via pathways.
- Explores uncommon signaling mechanisms for gene regulation.
- Three main sections: regulated proteolysis, direct interaction with transcription regulators, and
circadian rhythm control of gene expression.

*Regulated Proteolysis:*
- Notch receptor's crucial role in animal development and cell fate choices.
- Lateral inhibition process involving Delta signal protein.
- Notch, a single-pass transmembrane protein, undergoes successive proteolytic cleavages.
- γ-secretase mediates the final cleavage, associated with Alzheimer's disease.
*Glycosylation Regulation:*
- Notch and Delta are glycoproteins.
- Notch glycosylation influences their interaction and ligand specificity.
- Fringe family glycosyl transferases add extra sugars to Notch's O-linked oligosaccharide.

**Wnt Proteins and the Regulation of β-Catenin:**

*Introduction to Wnt Proteins:*

- Wnt proteins serve as local mediators and morphogens in development.
- Discovered independently in flies and mice.
- Humans have 19 different Wnt proteins with distinct functions.

*Wnt Signaling Pathways:*

- Two primary intracellular signaling pathways: Wnt/β-catenin and planar polarity.
- Wnt proteins initiate signaling by binding to Frizzled family receptors.

*Wnt/β-Catenin Pathway:*
- Focus on regulation of β-catenin involved in cell-cell adhesion and gene transcription.
- Degradation complex disruption allows β-catenin accumulation and nuclear translocation.
- β-catenin serves as a coactivator, inducing transcription of Wnt target genes.

**Hedgehog Proteins and Their Signaling Pathway:**

*Introduction to Hedgehog and Wnt Proteins:*

- Hedgehog and Wnt proteins are secreted signal molecules crucial in development.

*Hedgehog Proteins:*
- Active Hedgehog proteins covalently coupled to cholesterol and a fatty acid chain.

*Regulation of Cubitus interruptus (Ci):*

- Ci acts as a latent transcription regulator similar to β-catenin in the Wnt pathway.
- Proteolytic processing of Ci involves phosphorylation by PKA, GSK3, and CK1.

**NFκB-Dependent Signaling Pathway:**

*Introduction to NFκB Proteins:*

- NFκB proteins are latent transcription regulators found in most animal cells.
- Central to stressful, inflammatory, and innate immune responses, protecting multicellular
organisms from infection and injury.

*Activation of NFκB:*
- Various cell-surface receptors activate the NFκB pathway, triggering immune and inflammatory
responses.
**Nuclear Receptors and Ligand-Modulated Transcription Regulation:**

*Introduction to Nuclear Receptors:*

- Various small, hydrophobic signal molecules can diffuse across the plasma membrane and
bind to intracellular receptors acting as transcription regulators.

*Nuclear Receptor Superfamily:*

- These receptors are part of the nuclear receptor superfamily, structurally related.

*Function of Different Signal Molecules:*

- Steroid hormones influence metabolism and secondary sex characteristics.
- Vitamin D regulates calcium metabolism.
- Thyroid hormones enhance the metabolic rate of various cell types.

*Solubility and Transport:*

- Hydrophobic signal molecules bind to carrier proteins in the bloodstream and other
extracellular fluids for solubility.

*DNA Binding and Transcription Regulation:*

- Nuclear receptors bind to specific DNA sequences near target genes.
- Ligand binding alters the receptor's conformation, causing dissociation of inhibitory complexes
and binding of coactivator proteins, leading to gene transcription stimulation.

*A Global Environmental Signal:*

- Focus shifts to the regulation of gene expression by the light-dark cycle, a global
environmental signal resulting from the Earth's rotation.

Circadian Clocks and Negative Feedback Loops in Gene Expression Control :

Here are the concise bullet points for each section:

**Introduction to Circadian Clocks:**

- Internal circadian clocks exist in many organisms, regulating behaviors and physiological
processes.
- Anticipate environmental changes and reset by external cues like light.

**Cellular Basis of Circadian Clocks:**

- Circadian timekeepers are individual cells in multicellular organisms.
- In mammals, the suprachiasmatic nucleus (SCN) cells in the hypothalamus serve as central
timekeepers.
- SCN cells exhibit individual oscillations in gene expression with a ~24-hour period.

**Widespread Presence of Circadian Clocks:**

- Most mammalian cells have their own internal circadian rhythms.
- Various cell types in animals possess circadian clocks.

**Molecular Basis of Circadian Clocks:**

- Circadian clocks operate via negative feedback loops.
- A delayed negative feedback loop is central to circadian clock functioning.
- Transcription regulators inhibit their own transcription but with a time delay.
- Some cell types involve post-translational mechanisms in their circadian clocks.

**Reconstitution of a Cyanobacterial Circadian Clock with Three Proteins:**

- Synechococcus elongatus, a cyanobacterium, has a simple circadian clock with three proteins:
KaiA, KaiB, and KaiC.

**Core Oscillator Components:**

- KaiC is a central protein in the cyanobacterial clock, undergoing phosphorylation and
dephosphorylation in a 24-hour cycle.

**Interactions between Kai Proteins:**

- KaiA stimulates KaiC's autophosphorylation, and KaiB inhibits KaiA's stimulatory effect on
KaiC.

**Negative Feedback Loop:**

- The cyanobacterial circadian clock relies on a negative feedback loop involving Kai proteins.

**Reconstitution of Circadian Clock:**

- In a test tube, the three Kai proteins, when combined with ATP, reconstitute the circadian
clock's function.

**Light Entrainment:**
- The cyanobacterial circadian clock is entrained by the light-dark cycle, indirectly influenced by
light through changes in intracellular redox potential.