Cell Communication Types

• Cell communication involves signals traveling short or long distances.
• The receiving cell needs a specific receptor to detect a signal.

Types of Cell Communication

• Endocrine: Hormones travel through the bloodstream for long-distance communication.
• Paracrine: Signals act locally in the extracellular fluid, without entering the bloodstream for short-distance signalling.
• Synaptic/Neuronal: Electrical signals travel along nerve cell axons, releasing neurotransmitters to target cells directly and can be long distance communication.
• Contact-Dependent: A cell-surface signal molecule binds to a receptor on an adjacent cell through direct physical contact.

Comparing Communication Types

• Endocrine, paracrine, and neuronal signaling use similar signal molecules.
• They differ mainly in the speed and selectivity of signal delivery.

Cell Communication Analogy

• Endocrine: Like a radio announcement reaching many people over a distance, provided they have a radio.
• Paracrine: Like flyers posted in a local area, reaching those nearby.
• Synaptic/Neuronal: Like a letter sent to a specific person, potentially over a long distance.
• Contact Dependent: Like telling a friend something face-to-face.

Stimuli Recognized by Receptors

• Extracellular ligands
• Cell-to-cell contact
• Extracellular matrix

Extracellular Ligand Stimulus

• A ligand binds to a receptor on the cell surface.
• The receptor changes shape, altering its cytosolic tail.
• This change triggers intracellular changes; the ligand itself doesn't enter the cell.
• Example: insulin

Extracellular Matrix Stimulus

• A receptor binds to an extracellular matrix protein like collagen which alters the cytosolic tail.
• Binding initiates intracellular changes.

Cell-to-Cell Contact Stimulus

• Direct contact between two cells causes changes within the cells.

Similarities Among Stimuli

• All stimuli require receptors.
• They all induce changes inside the cells.
• The induced changes must be reversible.

Signal Initiation and Responses

• Receptor recognizes a stimulus.
• Signal is transferred to the cytoplasmic surface of the receptor.
• Signal is transmitted to an effector molecule.
• The response eventually ceases.

Signal Transduction

• It is the conversion of one signal type into another.
• An extracellular signal is converted into an intracellular signaling molecule.
• The extracellular signal doesn't enter the cell.

Signal Transduction Analogy

• Cell phones convert radio signals into sound signals, and vice versa.

Function Dependence

• The function of molecules depends on their shape.

Signal Transduction Pathway

• Primary transduction → relay → transduce and amplify → integrate → distribute.
• Most steps involve a molecule binding and changing shape, triggering a cascade effect.

Signal Transduction Pathway Explained

• An extracellular signal binds to a receptor, releasing a messenger into the cell.
• A secondary messenger is released by an activated effector.
• This messenger binds to a target protein, altering its shape and triggering further downstream effects.
• The process is a series of shape changes.

Advantages of Multi-Step Pathways

• Amplification: A single messenger can activate many downstream proteins.
• Integration: Different pathways can converge to activate the same target.
• Distribution: One protein can activate multiple different targets.

Signal Specificity

• The same signal molecule can induce different responses in different target cells.
• Example: Acetylcholine has same receptor structure on different cells, but different downstream effects
  • Heart pacemaker cell: slows heart rate
  • Salivary gland cell: releases enzymes
  • Skeletal muscle cell: causes contraction

Extracellular Signal Binding

• Extracellular signals bind to cell-surface receptors or intracellular receptors.
• Large, hydrophilic signals bind to cell-surface receptors, generating intracellular signaling molecules.
• Small, hydrophobic signals can cross the plasma membrane and activate intracellular enzymes or receptors, influencing gene transcription.
  • Examples: estrogen, cortisol, testosterone

Animal Cell Signals

• Animal cells require multiple signals:
  • Survival: continuous signals are needed by the cell to survive
  • Growth and division: additional signals may be needed for cell growth and division
  • Differentiation: additional signals are needed to change gene expression
  • Death: occurs in the absence of signals

Cancer Cells

• Cancer cells don't need continuous survival signals due to mutations that promote continuous division.

Speed of Extracellular Signals

• Extracellular signals can act slowly or rapidly.
  • Slow responses involve changes in gene expression and protein synthesis.
  • Rapid responses don't require gene expression changes as a signal can change a protein shape and function within milliseconds.
  • Example: signalling in the eye

Stimulus Reception

• The stimulus received by the cell-surface receptor is different than signal released in cell interior, the first messenger is converted into a second messenger.

Signal Pathways

• A series of distinct proteins alter the conformation of "downstream" proteins, leading to changes in gene expression.

Signaling Proteins

• Signal proteins relay, amplify, integrate, and distribute incoming signals.
  • Relay: carries the signal into the cell
  • Amplify: one upstream protein can modify and activate many copies of a downstream protein.
  • Integrate: convergence in pathway; Convergence of the pathways
  • Distribute: distribution of signal to other proteins
  • Scaffold proteins hold pathway proteins in proximity for faster and more efficient activation.

First and Second Messengers

• First messenger: the extracellular signal, which is generally hydrophilic.
• Second messengers: small, nonprotein intermediaries in signal transduction, with levels changing upon first messenger binding.
  • Examples: cAMP, calcium, lipid-derived molecules

Cyclic AMP (cAMP)

• As a secondary messenger it is released inside the cell to bind to targets to activate gene transcription.

Calcium

• As a secondary messenger it is kept at low concentration in the cytosol, stored in the ER and mitochondria.
• Increased cytosolic calcium acts as a signal.

Lipid-Derived Messengers

• Specialized phospholipids are activated and cleaved into messenger molecules.

Classes of Cell Surface Receptors

• Ion channel-coupled receptors
• G-protein-coupled receptors
• Enzyme-coupled receptors

Ion Channel-Coupled Receptors

• They open in response to extracellular signal molecule binding and are also called transmitter-gated ion channels.
• The signal molecule is distinct from the ion that passes through the channel.
  • Examples: neurotransmitters binding to receptors and acetylcholine and skeletal muscle receptors

G-Proteins

• When bound to GTP, they have a certain shape and activity.
• When bound to GDP, they have another shape and activity.

G-Protein-Coupled Receptors

• When a receptor binds to the extracellular signal molecule, the activated receptor signals to a G protein on the opposite side of the plasma membrane. The G protein activates or deactivates an enzyme or ion channel in the membrane.
• The G protein links the receptor and the effector.
• Receptor changes shape upon ligand binding, activating the G protein which then binds to the effector creating the secondary messenger.
  • Example: receptors in eye

Enzyme-Coupled Receptors

• When a receptor binds to an extracellular signal molecule, an enzyme activity is switched on at the other end of the receptor (inside the cell).
• Receptors may dimerize upon ligand binding. Some receptors have intrinsic enzymatic activity, while others associate with enzymes upon activation.

Molecular Switches

• The mechanism to turn on a protein, where many intracellular signalling molecules act as molecular switches.
• Proteins are switched "on" by modification and can be activated or inhibited by the addition or removal of a phosphate group or a GTP.
• Two types: Signalling by protein phosphorylation and signalling by GTP binding proteins

Molecular Switches: Protein Phosphorylation

• A shape change induced by phosphorylation.
• A protein kinase transfers a phosphate from ATP to a target protein, activating it.
• Protein phosphatase removes the phosphate, deactivating the protein.

Protein Kinase Pathway Example

• Inactive protein kinase (PK) 1 is activated upon ligand binding to its receptor.
• Active PK1 phosphorylates and activates PK2.
• Active PK2 phosphorylates and activates PK3.
• Active PK3 may activate a transcription factor, altering gene expression.

Protein Phosphorylation Details

• Phosphates are highly charged and can change a protein's shape and activity.
• Phosphorylation can either activate or inactivate a protein.
• Phosphorylation causes conformational changes.
• It may create a protein binding site.
• Occurs on serine, tyrosine, or threonine amino acids.
• Most pathway substrates are other enzymes.

Protein Kinases and Phosphatases

• They change the shapes/activities of the proteins they modify.
• There's a balance of kinase and phosphatase activities.
• The human genome encodes ~500 kinases and ~100 phosphatases.

Molecular Switches: GTP-Binding Proteins

• A GTP-binding protein is activated when it exchanges its bound GDP for GTP and is switched off when hydrolyzing GTP to GDP by the protein GTPase activity.
• There is a signal receptor that tells when GDP has to be exchanged for GTP

Regulation of Monomeric GTP-Binding Proteins

• These proteins are controlled by two types of regulatory proteins:
  • GAP - GTPase-activating proteins
  • GEF - Guanine nucleotide-exchange factors

Guanine Nucleotide-Exchange Factors (GEF)

• GEFs remove GDP from the protein, facilitating GTP binding to switch the GTP-binding protein on.

GTPase-Activating Proteins (GAP)

• GAPs stimulate GTP hydrolysis to GDP, switching the GTP-binding protein off which causes the protein to rapidly hydrolyze GTP back to GDP.

Alternate Signal Transduction Pathways

• G protein-linked receptor or G-protein coupled receptors (links receptor to the effector which will lead to a series of events).
• Protein kinase receptor (enzyme will phosphorylate itself leading to a series of events).

G-Protein-Coupled Receptors (GPCRs)

• A receptor will bind its ligand which causes effector to release a secondary messenger.
• It is the largest superfamily of proteins in animal genomes.
  • Example: nematode worm has 19,000 genes of which 1000 are GPCRs.
• Target of ~40% of modern medicinal drugs.

GPCR structure

• All have 7 transmembrane alpha helices.
• The ligand binds to external face, changing the cytosolic tail.
• The cytosolic side binds to a G protein inside the cell.

GPCR Mechanism Overview

• Extracellular signal molecule actives receptor.
• Inactive G protein gets activated by the receptor, GDP is exchanged for GTP.
• Activated alpha and beta-gamma subunits of G protein split
• Activated subunits activate (or inactivate) target proteins.
• Activated alpha subunit hydrolyzes GTP to GDP and becomes inactive.
• Inactive alpha subunit reassociates with beta-gama subunit, reforming inactive G protein.
• G protein is ready to couple to another activated receptor (can bind multiple G-proteins at a time).

How GPCRs activate G-proteins

• Activated GPCRs activate G proteins by encouraging the alpha subunit to expel its GDP and pick up GTP.
• Binding of an extracellular signal molecule to the receptor changes the conformation of the receptor, which in turn alters the conformation of the bound G-proteins.
• The alteration of the alpha subunit of the G protein allows it to exchange its GDP for GTP.
• This exchange triggers an additional conformational change that activates both the alpha subunit and a beta-gamma complex.
• The beta-gamma complex can dissociate to interact with their preferred target proteins in the plasma membrane or it can remain with the alpha subunit and activate proteins.

GPCR messengers

• First messenger: ligand that binds to receptor
• Second messenger: effector that gets activated

Effector Function

• Is an enzyme that will activate another messenger in the cell.
• Can be activated by the alpha subunit or the beta-gamma subunits of the G protein.
• Does not directly interact with the receptor, G protein links them.

G-protein Subunits and Lipid Anchors

• Both alpha and gamma subunits have covalently attached lipid molecules for anchoring to the plasma membrane.

GPCR: Signaling relay to effector

• The activated α subunit changes the effector shape to its “on” position and activates it.

GPCR: Ending the response

• GTP of α subunit is hydrolyzed to GDP and α subunit changes shape, becoming inactive.
• G protein no longer binds effector, but reforms trimer with beta-gamma subunit.
• Effector changes shape and becomes inactive.
• Desensitization of the receptor also serves to end the response, inactivates receptor even if ligand is still bound.

Receptor Desensitization Steps

• GRK (G protein-coupled receptor kinase) phosphorylates the tail of receptor (part of receptor that binds G protein) using ATP.
• Arrestin recognizes and binds to the phosphorylated receptor blocking G protein binding thus the receptor loses its ability to bind to other G-proteins.
• Receptor endocytosed from PM.

GPCR, Effector, and Secondary Messenger Example

• GPCR: glucagon receptor
• Effector: adenylyl cyclase
• Secondary messenger: cAMP

Specificity of G Protein-Coupled Responses

• Not all parts of signal transduction machinery identical in all cells.
• Multiple forms of receptors (9 different isoforms of epinephrine receptors) with different ligand and G protein affinities.
• Multiple G proteins (20 Gα; 5 Gβ; 11 Gγ ); various combinations.
• Can be stimulatory or inhibitory.
  • Example: Gαs stimulatory and Gαi inhibitory

GPCRs and Ion Channels

• Some G proteins directly regulate ion channels, making the ion channel the effector of the GPCR.
• Example: A Gαi (inhibitory) protein directly couples receptor activation to the opening of K+ channels in the plasma membrane of the heart pacemaker cells:
• Binding of the neurotransmitter acetylcholine to its GPCR on the heart cells results in the activation of the G protein, Gαi →activation of alpha changes GDP to GTP.
• The alpha does not bind to the beta-gamma complex beta gamma complex is activated (beta-gamma does not bind GDP or GTP but seems to be inactivated when associated with the alpha)
• When K+ channels open, K+ leaves the heart and the heart is hyperpolarized which then slows down the heart rate.
• inactivation of the alpha subunit by hydrolysis of its bound GTP returns the G protein to its inactive state, allowing the K+ channel to close

Enzymes as effectors for GPCRs

• Some G proteins activate membrane-bound enzymes.
• Enzymes activated by G proteins catalyze production of small intracellular signaling molecules.
• Ligand binds to the receptor and the G protein will activate an enzyme.
• Enzymes activated by G proteins catalyze production of small intracellular signaling molecules.
  • Examples: Adenyl cyclase and phospholipase C
  • Secondary messengers: cAMP; IP3/DAG
• One common system involves the G protein activating enzyme effector adenyl cyclase that will make lots of cAMP (cyclic AMP) which will diffuse throughout the cell and activate other things.

Glucose Regulation

• Glucose is stored in animal cells as glycogen (polymer of glucose).
• When the body needs glucose, glycogen is broken down back into glucose.
• When stored glycogen in the liver is low, glucagon and epinephrine are released to make more glucose.
• Glucagon and epinephrine release activates enzyme called phosphorylase (glycogen phosphorylase makes more glucose).
• Phosphorylase carves off one glucose from glycogen and makes it glucose 1 phosphate which will be used for something else in the cell.
• When there is too much glucose in the blood, the body releases insulin which causes glucose to be taken up in the cell and put in the storage form of glycogen via glycogen synthase.

Glucagon and Epinephrine Function

• They regulate glucose levels and phosphorylase as well as glycogen synthase.
• They increase blood glucose by inhibiting glycogen synthase and activating phosphorylase.
• Glucagon is released by the pancreas.
• Epinephrine (adrenaline) is released by the adrenal glands.

Glucagon, Epinephrine & Adenylyl Cyclase

• Glucagon and epinephrine bind to different receptors but lead to the same intracellular response as they both activate adenylyl cyclase. They both merge in the same downstream pathway using the same secondary messenger cAMP.

Glycogen Breakdown/Synthesis

• Phosphorylase: enzyme that breaks down glycogen making more glucose.
• Glycogen synthase: enzyme that takes excess glucose and stores it as glycogen.

Muscle Cells and Glucose

• Muscle cells need sugar and lots of energy so they will go into glycolysis instead to produce ATP.

cAMP Formation & Degradation

• It comes from the effector Adenylyl cyclase: cyclic AMP is synthesized by adenylyl cyclase and degraded by cyclic AMP phosphodiesterase.
• cAMP is formed from ATP by a cyclization reaction that removes two phosphate groups from ATP and joins the free end of the remaining phosphate group to the sugar of the AMP molecule.
• The degradation reaction breaks the bond between the phosphate and the sugar of the AMP, forming AMP.

Adenylyl Cyclase

• Integral membrane protein that makes cAMP and is an effector.

Adrenaline and Glycogen Breakdown

• Adrenaline stimulates glycogen breakdown in skeletal muscle cells:
  • Adrenaline (hormone) activates a GPCR, which turns on a G protein (Gs→G stimulatory) that activates adenylyl cyclase to boost the the production of cyclic AMP.
  • The increase in cyclic AMP activates Protein Kinase A (PKA), which phosphorylates and activates an enzyme called phosphorylase kinase (PKA is activated by cAMP).
  • Phosphorylase kinase activates glycogen phosphorylase (enzyme that breaks down glycogen to release glucose).

Ways to Increase Glucose in the Blood

• Increasing the breakdown of glycogen.
• Inhibiting the formation of glycogen.
• Activating enzymes needed to make glucose from scratch
• All done by PKA which is activated by the increase in cAMP which resulted from the activation of glucagon or epinephrine.

cAMP Formation

• Glucagon and epinephrine bind to a GPCR.
• G protein gets activated and the alpha subunit will go over and bind to adenylyl cyclase (the effector).
• Activated adenylyl cyclase will turn ATP into cAMP by removing two phosphates.
• cAMP acts as a secondary messenger and activates other things.

Protein Kinase A (PKA)

• An enzyme that is activated by cAMP
• Activates phosphorylase kinase.
• Also phosphorylates glycogen synthase, which inhibits glycogen synthesis (phosphorylation is going to inhibit the glycogen synthase).
• Kinases phosphorylate their targets.

Adrenaline and Glucose Synthesis

• Binding of the signal molecule to the GPCR can activate adenylyl cyclase and increase cAMP.
• Increase in cAMP activates PKA.
• PKA moves into the nucleus and phosphorylates specific transcription regulators.
• Stimulate the transcription of a whole set of target genes, as well as activating gene transcription.
• Enzymes activated in the target gene are ones needed for gluconeogenesis (generation of glucose).

Signal Amplification

• Each GPCR activates multiple G proteins.
• Each active adenylyl cyclase make many cAMPs.
• Each active PKA can phosphorylate multiple phosphorylase kinases.

Reversal of Signal

• Pathways cannot stay on forever and must be turned off.

Reversal of Signal: Glucose Pathway

• Phosphatase-1 removes all of the phosphates that were added by PKA in every step of the pathway.
• cAMP is destroyed by cAMP phosphodiesterase (cAMP becomes AMP) meaning cAMP can no longer turn on PKA.
• GPCR can get turned off by a kinase and then arrestin will bind to the receptor, keeping it from being able to activate more G proteins.
• Inactive G proteins will not be able to activate the effector so the effector will be turned off as well.

Phosphatidylinositol (PI) and Phosphoinositides (PIPs)

• They contain inositol and are used for signaling.
• PIP results from the phosphorylation of OH in inositol of PI usually the 3, 4, or 5 carbon get phosphorylated.
• Cause proteins to come up and dock to the membrane.

PI and PIP Naming

• Named according to ring position and number of phosphate groups.
• Phosphate that is part of the phosphotidylinositol does not get included in the numbering of the phosphates only the phosphates that were added by a kinase are included.
• Example: PI(3,4)P2: (3,4) means that carbon 3 and 4 are phosphorylated and the 2 means that only 2 carbons were phosphorylated.

2/6 Carbons of Inositol

• Carbons 2 and 6 on inositol usually don't get phosphorylated when PI is becoming PIP due to sterol reasons; carbons 2 and 6 are closer to the ring.

PI Kinases and Phosphatases

• Animal cells have several PI and PIP kinases and phosphatases.
• There are many different ways, orders, and sequences to phosphorylate the PI.
• Kinases will add the phosphates and the phosphatases will remove them.
• Kinases that add the phosphates will be activated by the cell signalling pathway.

PIs and Proteins

• PI headgroups are recognized by protein domains that discriminate the different forms.
• Proteins are recruited to regions of the membrane where these PIs are present.
• Proteins were soluble in the cell and the protein came up and docked on the PI headgroup in the membrane. which will then interact with it

Phospholipase C

• GPCR enzyme effector
• Membrane bound protein.
• Not active until an activated G protein activates it.
• Releases a second messenger when activated the second messenger is IP3/DAG.
• It is an enzyme that cuts the bond between the phosphate and the glycerol in the phosphotidylinositol, releasing the phosphate of the headgroup.

Phospholipases

• They are hydrolytic enzymes that split phospholipids.
• There are three types: PLA -- phospholipase A, PLD -- phospholipase D, PLC -- phospholipase C

Phosphatidylinositol (PI)- Mediated Responses

• Phospholipase C cleaves PIP2 into DAG (diacylglycerol) and IP3.
• DAG (glycerol + 2 fatty acid tails): hydrophobic and stays in the membrane which will signal some things and is a secondary messenger.
• IP3 (Inositol head and the phosphate): hydrophillic and moves further into the cell for signalling and is a secondary messenger.

Phospholipase C (PLC) Activation

• Gets activated by a signaling molecule:
• Receptor binds its ligand and activates the G protein.
• Alpha subunit of G protein activates PLC.
• Activated PLC will cut the bond in the PI and will release the IP3 which is soluble and hydrophilic and will go throughout the cell.

Diacylglycerol (DAG)

• Glycerol with the two fatty acid tails.
• Lipid molecule remains in Plasma Membrane.
• Recruits and activates protein kinase C (PKC).

Protein Kinase C (PKC)

• Activated by DAG.
• Serine/threonine kinase.
• Important in many cellular events to include (e.g., cell growth, differentiation, metabolism and transcriptional activation).

Phorbol Esters

• Plant compounds that mimic DAG and activate PKC and the cells lose growth control and behave as malignant cells.

Consequences of DAG Addition

• Take a cell that does not grow or divide and add something that mimics DAG and the cells start to divide uncontrollably (lose growth control).

Researching DAG

• Artificially turn DAG on, by using something that mimics DAG.

IP3 (inositol 1,4,5-trisphosphate)

• Small, water soluble (hydrophilic).
• Binds to a Ca++ channel receptor on smooth endoplasmic reticulum (SER).
• Releases Ca++ from the SER.
• Goes to the ER to release Ca++ after it is cut off and released from the membrane.

PLC Activation of Two Signaling Pathways

• Activated alpha and beta-gamma subunits activate PLC.
• Activated PLC hydrolyzes inositol phospholipid which produces two small messenger molecules: IP3 and DAG.
• IP3 diffuses through the cytosol and triggers the release of Ca++ from the ER by binding to and opening Ca++ channels in the ER membrane-→electrochemical gradient causes Ca++ to rush out of the ER and into the cytosol.
• DAG remains in the plasma membrane and together with the Ca++ help activate PKC.
• PKC phosphorylates and activates its own set of intracellular proteins.

Calcium as an Intracellular Messenger

• Low Ca++ in the cytosol with high Ca++ in the ER.
• Ca++ release from intracellular stores acts as a second messenger.
• Can act as the first messenger for hormones, neurotransmitters, electrical activation (muscle).