15c Intracellular Components

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Signal Transduction: Most intracellular pathways contain multiple steps; Message gets converted from one form into another...

It is the process by which a chemical or physical Signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation catalyzed by protein kinases, which ultimately results in a cellular response.

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Three Classes of Intracellular Signaling Components:

Kinases: Signaling by phosphorylation;

G Proteins: Signaling by G-Protein (in)activation;

The Second Messengers.

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[1] Kinases - Attach a (P) to a protein from ATP

Kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the substrate gains a phosphate group and the high-energy ATP molecule donates a phosphate group.

The phosphate group can be removed by the Phosphatase - back to resting state.

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Phosphorylation on hydroxyl groups

- In Eukaryotes, the phosphate group usually attaches to Amino Acids with a hydroxyl group (-OH) in the side chain: Serine, Threonine;?Tyrosine (aromatic ring).

- Two classes of kinases: Serine / Threonine Kinases; Tyrosine Kinases

- Different Kinases recognize and phosphorylate different sets of target proteins. The kinase recognizes both the amino acid to be phosphorylated and also other nearby amino acids?(Kinase Recognition Sequence) in the target; subclasses of Ser/Thr or Tyr Kinases?have different Recognition Sequences.

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Signaling by Phosphorylation (Left Picture)

Activation: Active kinase, Low-level phosphatase - Kinase wins;

Inactivation: - Inactive kinase, Low-level phosphatase - Phosphatase wins.

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[2] G Proteins (Recall p. 14)

G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switches inside cells, and are involved in transmitting Signals from a variety of stimuli outside a cell to its interior. Their activity is regulated by factors that control their ability to bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When they are bound to GTP, they are turned “on,” and, when they are bound to GDP, they are turned “off.” G proteins belong to the larger group of enzymes called GTPases.

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GEFs, Guanosine nucleotide Exchange Factors, are proteins or protein domains that activate monomeric GTPases by stimulating the release of GDP to allow the binding of GTP;?GDP must be released to bind new GTP.

GTPase-activating proteins or GTPase-accelerating proteins (GAPs) are a family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the Signaling event. GAPs are also known as RGS (Regulators of G protein Signaling) proteins.

G protein alone (green in the top picture): slow intrinsic rate of hydrolysis / slow intrinsic rate of GDP release; GEFs and GAPs accelerate these rates.

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Passing the message:

GEFs and GAPs receive many messages from upstream molecules to turn the G proteins “on” or “off.” Then, G-proteins bind downstream target proteins to activate or inhibit them - G proteins diffuse laterally.

G-proteins: Lipid-linked in the plasma membrane

G-protein targets: in the plasma membrane

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[3] Second Messengers

Second messengers are intracellular Signaling molecules released by the cell in response to exposure to extracellular Signaling molecules -- the first messengers.

Second messengers are not proteins; they are small, mobile, intracellular, allosteric regulators.

Examples of second messenger molecules include cyclic AMP (cAMP), cGMP, inositol trisphosphate (IP3), diacyl glycerol (DAG), and calcium (Ca2+).

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a. Adenylyl Cyclase & cAMP

Adenylyl cyclase is a transmembrane protein that works as an enzyme to synthesize cyclic AMP from ATP. Cyclic AMP allosterically activates downstream targets, functions as a second messenger to relay extracellular Signals to intracellular effectors, particularly Protein Kinase A?(PKA). cAMP phosphodiesterase (PDE) can inactivate cAMP, see next page top left picture.

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b. IP3?& DAG

PIP2, phosphatidylinositol 4,5-bisphosphate, see the top right picture. PIP2?can be cleaved to form two second messengers: IP3?and DAG. Phospholipase C (PLC) cleaves PIP2?? DAG + IP3. DAG has two lipid tails?(embedded in the membrane).

IP3?is made by hydrolysis of PIP2?by PLC. Together with DAG, IP3?is a second messenger molecule used in Signal transduction in biological cells. While DAG stays inside the membrane, IP3?is soluble and diffuses?through the cell, where it binds to its receptor, which is a calcium channel located in the ER. When IP3?binds its receptor, calcium is released into the cytosol, thereby activating various calcium-regulated intracellular Signals.

Eg Inactivation: DAG? 2 HC chains + glycerol

IP3?? inositol + 3 Pi

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c. Calcium Ions

Calcium in the cell is stored in the ER. It has very low concentration in the cytoplasm. The Signal transduction pathway can cause the Ca2+-release channel (right picture) in the ER to open up, to release Calcium ions from the ER down the concentration into the cytoplasm, where Calcium can bind & allosterically activates different target proteins.

When the upstream Signal stops, the channel will close again. But there are some Calcium ions in the cytoplasm. So there is an ATPase (left picture) constitutively & actively pumps Calcium to ER.

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15d Why Long Intracellular Signaling Pathways?

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Signaling pathway exist that have only one intracellular component (right): steroid hormones.

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Typical Signaling paths do have many, many steps (left):

Extracellular Signal?activates TM receptor;

Activated receptor?activates G protein;

Activated G protein?activates adenylyl cyclase;

Activated adenylyl cyclase?makes cAMP;

cAMP activates Protein Kinase?A;

Kinase enters nucleus, phosphorylates a transcription factor, causing changes in gene expression.

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If a one-step Signaling pathway worked for steroid hormones, why would a cell use a more complicated, multi-step Signaling pathway?

Recall metabolism: Metabolism uses?multi-step pathways so energy could be released in small bits and captured in a single ATP, NADH, or H+ pumped across a membrane. In Signaling, the purpose of multi-step pathways is different.

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Four general advantages of multi-step pathways in Signaling:

[1] Integration of Multiple Signals

Each cell may not just receive one Signal from the environment, there may be many different Signals in different combinations. The cell needs to combine all different Signals together to decide how to respond.

No Signals = cell dies (apoptosis, programmed cell death). Cancer cells may not understand the “l(fā)ocal languages / Signals” - lead to?the?death of them.

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[2] Signals are relayed from the membrane

Signals are relayed / moved from the membrane where they are first received to the interior of the cell through multi-step pathways.

Example (right picture): extracellular Signal first detected on the membrane - synthesizing cAMP which is diffusible to anywhere in the cell - affect behaviors of other proteins (eg TF, nucleus)...