Chapter 15 Signal Transduction and G Protein- Coupled Receptors

Bolded terms and pathways from slides

Cell-adhesion molecules mediate signal transduction

Can respond to changes in the ECM, tansduce signals, and promote altered cell activity

regulate:

• migration

• proliferation

• apoptosis

• enzymatic activities

• gene transcription

Receptor Protein

A protein that binds a ligand, which could be a small molecule such as adrenalin, a growth factor such as epidermal growth factor, a hormone such as insulin, or another macromolecule, usually with engagement of a signaling cascade as a consequence.

Ligands

Signaling molecules that bind specific receptors triggering a specific cellular response

Any molecule, other than an enzyme substrate, that binds tightly and specifically to a macromolecule, usually a protein, forming a macromolecule-ligand complex.

Agonist

A molecule, often synthetic, that mimics the biological function of a natural molecule (e.g., hormone)

Activating

Antagonist

A molecule, often synthetic, that blocks the biological function of a natural molecule (e.g., hormone)

Blocking

Signal Transduction

The process by which cells receive and respond to external signals.

Involves change in the position and type of information transmitted through a cell.

Signal Transduction Pathway

The set of proteins or small molecules involved in relaying a chemical message from the receptor at the cell surface to the target molecule(s) inside the cell.

Second messengers

Small, non-protein, intracellular molecules that relay, amplify, and transduce signals

Transduction enzymes

Highly specific promote transduction through allosteric regulation of the next protein

Transduce signaling via covalent modifications

• kinases

• phosphatases

• cleavage enzymes

• enzymes that catalyze synthesis of second messengers

Effector Proteins

An enzyme, transcription factor, or cytoskeletal protein whose activation leads directly to changes in cellular activities.

Transduction regulation of effector proteins

Transduction pathways regulate terminal effector proteins.

• Enzymes that alters cell behavior or physiology- rapid response

• specific transcription factors that alters gene expression- slower long-term response

• Often a combination of both

Specific transcription factors

Bind regulatory DNA sequences either in the promoter or discrete regulatory DNA regions (cis-regulatory elements)

Binding transcription factors to these elements can either activate or inhibit RNA polymerase and therefore regulate gene transcription.

Enhanser

A regulatory sequence in eukaryotic DNA that may be located at a great distance from the gene it controls or even within the coding sequence. Binding of specific proteins to an enhancer modulates the rate of transcription of associated gene.

Cis-Regulatory elements (CREs)

discrete regulatory DNA regions

Binding transcription factors to these elements can either activate or inhibit RNA polymerase and therefore regulate gene transcription.

Feedback inhibition

The final effector protein also blocks continued transduction of the pathway- ensuring a transient, but appropriate amount of response.

Endocrine Signaling

Ligands called hormones are secreted by specialized cells of the various endocrine glands and travel through the circulation system to act on target cells in distant tissues

Examples:

• insulin/glucagon

• testosterone/estrogen

• cortisol and thyroid hormones

Some ligands can act as both endocrine and paracrine signals. Epinephrine, for example, acts hormonally as part of the fight or flight response, but is also a neurotransmitter.

Hormone

Generally any extracellular substance that induces specific responses in target cells; specifically, those signaling molecules that circulate in the blood and mediate endocrine signaling.

Paracrine Signaling

Secreted ligands diffuse a short distance through the extracellular matrix before binding receptors on nearby target cells (1 to 20 cells away)

Examples:

• Neurotransmitters that signal adjacent, but unconnected cells at their synapse

• Diverse growth factors that control rates of proliferation and or differentiation: immature cells adopting terminal fates

Some ligands can act as both endocrine and paracrine signals. Epinephrine, for example, acts hormonally as part of the fight or flight response, but is also a neurotransmitter.

The secreted ligands diffuse from the source and generate a concentration gradient.

Growth Factors

An extracellular polypeptide molecule that binds to a cell-surface receptor, triggering an intracellular signaling pathway generally leading to cell proliferation

Autocrine Signaling

Synthesis and secretion of ligands that sustain some physiological activity of the cell itself

• Most autocrine ligands are growth factors

• For example, the constant replacement of skin cells is driven by proliferation induced via autocrine signaling

• May types of cancer overproduce growth factors, enhancing their growth rate

Juxtacrine Signaling

Both the receptor and ligand are membrane bound

• Therefore, signaling only occurs between immediately adjacent cells

Signal transduction

The conversation of an impulse or stimulus from one physical or chemical form to another; the processes by which cells respond to extracellular signals.

The activated receptor relays this information to cytoplasmic peoteins and other molecules that:

• Amplify the signal

• Integrate it with other pathways

• Generate a cell response

Lipid derived hormones

enter cells, via endocytosis or diffusion

cytosolic receptors

Some ligands-like lipid derived hormones- enter cells, via endocytosis or diffusion. They bind and acrivate soluble cytosolic receptors.

These cytosolic receptors are usually specific transcription factors.

Cell-Surface Receptors

Protein embedded in the plasma membrane that has an extracellular domain that binds an extracellular molecule(s), called a ligand. Many cell-surface receptors bind to extracellular signaling molecules; such binding includes conformational changes in the receptor, altering the activity of the receptor’s intracellular domain, which transmits the signal to the interior of the cell.

Basal State

When ere is no signal cells capable of receiving that signal are said to be a basal or unstimulated state. A condition in which cells perform only basic, housekeeping functions, common to all cells of that type.

Principles of signal transduction

The same ligand may activate different responses in different cells because:

• They bind different receptor on the cells

• They bind the same receptor, but it activates a different transduction pathway

• Expression of different regulators of a shared pathway

• Expression of different terminal effector proteins

• Expression of different targets of a shared effector protein

• Different combinations of ligands activating different combinations of pathways

Mechanisms of signal transduction:

1. Some activated cell-surface receptors directly regulate a cytoplasmic enzyme that catalyzes synthesis of second messengers.

2. Other cell-surface receptors recruit cytoplasmic proteins to their intracellular domains and initiate a series of protein-protein interactions that transduction the signal

   • often mediated by kinase proteins

In both cases, signal transducction leads to the activation of one, or a small number of, specific effector proteins.

Effector proteins general functions

1. Enzymes that immediately alter some cellular process by regulating a key event or

2. Transcription factors that translocate to nucleus and regulate expression of specific target genes

Switches

Two major types of switches:

1. Those that regulate phosphorylation state:

   • Kinases and Phosphatases

2. G proteins that allosterically regulate targets proteins. Two families:

   • Small monomeric G proteins- we’ve seen several examples of these

   • Heterotrimeric G proteins- the focus of CH 15

Phosphorylation

The covalent addition of a phosphate group to a molecule such as a sugar or a protein. The hydrolysis of ATP often accompanies phosphorylation, providing energy to drive the reaction and the phosphate group that is covalently added to the target molecule. Enzymes that catalyze phosphorylation are called kinases.

Protein kinase

An enzyme that chemically adds a phosphate group, usually the gamma phosphate from ATP, to serine, threonine, or tyrosine reside of another protein.

Protein phosphatase

An enzyme that chemically removes a phosphate group from another protein by hydrolysis.

Monomeric G protein

A monomeric GTPase with a structure similar to that of the Ras protein that changes conformation when a bound GTP is hydrolyzed to GDP and phosphate

Heterotrimeric G proteins

A class of GTPase switch proteins, composed of alpha, beta, and gamma polypeptides, that bind to and are activated by certain cell-surface receptors. When activated, hertotrimeric G proteins release GDP and bind GTP.

GTPase switch proteins cycle between active and inactive state

GTPase protein superfamily members often serve as signal transduction on-off switches:

• ON conformation- GTP bound

• OFF conformation- GDP bound

  • GEFs (guanine nucleotide exchange factors) catalyze dissociation of bound GDP and replacement by GTP- active G proteins

  • GAPs (GTPase-activating proteins; aka regulators of G protein signaling) catalyze hydrolysis of GTP to GDP and therefore promote inactivation of G proteins

  • Guanine nucleotide-dissociation inhibitors (GDIs)- prevent G proteins from exchanging GDP for GTP and therefore keep G proteins inactive

G protein- coupled receptor (GPCR)

Member of a large class of cell-surface signaling receptors, including those for epinephrine, glucagon, and yeast mating factors. All GPCRs contain seven transmembrane alpha helices. Ligand binding leads to activation of a coupled trimeric G protein, thereby initiating intracellular signaling pathways.

G_⍺ subunit

The catalytic G-protein that is regulated by its GTP/GDP bound state. Homologous to the small monomeric G proteins we have already discussed. All G_⍺ subunits are lipid anchored and remain bound to the membrane even after activation.

G_β𝛾 subunit

A dimer that binds G_⍺- GDP and keeps it inactive until a GPCR activates it. The G_𝛾 subunit is also lipid anchored so G_β𝛾 also remains membrane tethered even after separation from G_⍺ . G_β𝛾 & G_⍺ united unless inactive.

Generalized GPCR signal transduction pathway

1. Ligand binds GPCR, altering its conformation and ability to engage heterotrimeric G-protein

2. Bound receptor activates hetero-trimeric G protein, via catalyzing ⍺-subunit exchange of GDP for GTP

3. ⍺-subunit disengages from β𝛾 dimer

4. ⍺-subunit binds and regulates activity of a target enzyme

5. β𝛾 dimer also usually has regulatory targets- like an ion channel shown below

The enzyme regulated by G_⍺-subunits regulates production of a second messenger

Some G_⍺-subunits are positive activators of their target effector enzymes, other inhibit second messenger synthesis

The major G_⍺ families and their target effector enzymes are:

• G_⍺s: Stimulatory- Activate the effector enzyme adenylyl cyclases (cAMP)

• G_⍺i: Inhibitory- Inhibit the effector enzyme adenylyl cyclase (cAMP)

• G_⍺q: Activate the effector enzyme phospholipase C

• G_⍺t: Activate the effector enzyme phosphosiesterase

cAMP

Generated from ATP by the enzyme adenylyl cyclase

Allosteric activator of the enzyme Protein Kinase A

¼ common intracellular second messengers

cGMP

Generated from GTP by the enzyme guanylyl cyclase

Allosteric activator of the enzyme Protein Kinase G and specific cation channels

¼ common intracellular second messengers

cyclic guanosine monophosphate

A second messenger that opens cation channels in rod cells and activates protein kinase G in vascular smooth muscle and other cells.

Adenylyl cyclase

One of the several enzymes that is activated by binding of certain ligands to their cell-surface receptors and catalyzes formation of cyclic AMP (cAMP) from ATP; also called adenylate cyclase.

Phospholipase

One of serval enzymes that cleave various bonds in the hydrophilic end of phospholipids.

Phospholipase C

An effector enzyme which catalyzes cleavage of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) into the two molecules:

• inositol 1,4,5-triphosphate (IP₃) (second messenger)

• Diacylglycerol (not considered a second messenger b/c stuck in the membrane)

Inositol 1,4,5-triphosphate (IP₃)

Functions as a second messenger. It diffuses into the cytoplasm and binds the IP₃ receptor on ER membrane

¼ common intracellular second messengers

• The IP₃ receptor is a lignad-gated Ca²⁺ channel- IP₃ binding ops the channel and releases Ca²⁺ into the cytoplams

• Ca²⁺ bind and regulates several Ca²⁺ dependent proteins

Calcium ions ( Ca²⁺ )

Released from the ER, or imported from extracellular space:

¼ common intracellular second messengers

Activates:

• Calmodulin, a ubiquitous switch protein

• A subset of protein kinase C proteins (PKC)

• Diverse other regulatory proteins (troponin in muscle cells, for example)

1,2-diacylglycerol (DAG)

remains membrane associated- so it is not a true second messenger

• it cooperatively activates with Ca²⁺ a subset of protein kinase c proteins

Epinephrine

regulates body’s fight or flight stress response pathway

A catecholamine secreted by the adrenal glad and some neurons in response to stress; also called adrenaline. It functions as both a hormone neurotransmitter, mediating fight-or-flight responses (e.g., increased blood glucose levels and heart rate)

Glucagon

expressed in response to low blood sugar level

A peptide hormone produced in the cells of pancreatic islets that triggers the conversion of glycogen to glucose by the liver; acts with insulin to control blood glucose levels.

cAMP phosphodiesterase (PDE)

This enzyme is activated by other pathways to stop this transduction. It catalyzes hydrolysis of the cyclic bond-releasing AMP

Protein Kinase A (PKA)

cAMP allosterically activates Protein Kinase A (PKA)

A family of soluble serine/threonine kinases

• two catalytic kinase subunits catalyze transfer of terminal phosphates from ATP to hydroxyl groups of specific Serine or Threonine residues on target proteins

• two regulatory subunits (inhibitory) whose conformation is altered by cAMP

• cAMP activates protein kinase A by negative allosteric regulation of the regulatory subunits

cAMP regulates diverse activities depending on:

• the cell type in which cAMP is produced

• which PKA is expressed

• which PKA target proteins are expressed

different cell types express different PKAs and PKA targets- this is what is responsible for distinct cellular responses.

GPCR pathway

G protein coupled receptor pathway

signal transduction pathways initiated by the binding of a ligand to a GPCR, triggering a cascade of events that ultimately lead to a cellular response, often involving G proteins and second messengers.

G_⍺s flowchart

ligand → GPCR → G_⍺s (stimulatory) → Adenylyl cyclase → +ATP cAMP → PKA activation by binding and inhibiting PKA negative regulatory subunits → PKA phosphorylates cell-type specific target proteins to generate appropriate response

G_⍺i flowchart

ligand → GPCR → G_⍺i (inhibitory) → adenylyl cyclase → cAMP synthesis blocked

Enzymatic response

Inhibition of glycogen formation and glycogenolysis- hydrolysis of glycogen to G6P

½ responses that lead to elevated Glucose-6-phosphate (G6P) to aid in ATP synthesis

• active adenylyl cyclase synthesizes cAMP from ATP which binds and activates PKA

• PKA phosphorylates and inactivates glycogen synthase halting glucose polymerization

• PKA also phosphorylates and activates glycogen phosphorylase kinase which in turn phosphorylates and activates glycogen phosphorylase - glycogen is cleaved into glucose-1-phosphate monomers which can be converted into glucose-6-phosphate for immediate use in muscles, while a liver enzyme removes the phosphate to the sugar can be released into the blood stream

• PKA promotes glycogen hydrolysis by regulating activity of an additional enzyme called phosphoprotein phosphatease (PP)- a protein that removes phosphate groups from residues on target proteins

  • PP removes phosphates from GPK and GP, thereby inhibiting these proteins and blocking glucose release

  • PP removes phosphates from Glycogen synthase- which enhances its activity and therefore increases glycogen production

activated PKA phosphorylates and activates an inhibitor of PP, leading to phosphorylation and activation of GPK and GP and the inhibition of GS

Transcriptional response

Transcription of gluconeogenesis genes that promote the synthesis of glucose from precursor metabolites like lactate and pyruvate- the reverse of glycolysis.

This includes genes encoding the three enzymes that catalyze the irreversible steps of glycolysis are turned off and genes encoding enzymes that catalyze the reverse reactions are transcribed.

½ responses that lead to elevated Glucose-6-phosphate (G6P) to aid in ATP synthesis

• PKA is also transported into the nucleus, where it controls expression of specific genes- in the liver, those invloved in gluconeogenesis

• CRE: cAMP Response Element- regulatory DNA (enhansers) adjacent to genes activated by PKA

• CREB: cAMP Response Element Binding Protein- transcription factor that binds CRE and activates expression of associated genes

• CBP/P300: CREB binding protein- links CREB to RNA polymerase promoting transcription.

• PKA phosphorylates and activates CREB, leading to assembly of the RNA transcription complex and activation of gene expression

• The same transcription factor CREB is activated by PKA is diverse cell types downstream of cAMP and different GPCRs

• Different sets of target genes are transcribed in these different cell types because of chromatin structure and the accessibility of genes for transcriptional regulation

Gluconeolysis

Major way glucose is made avaliable to the body when blood sugar levels are low (glucagon) or when excited/ stimulated (epinephrine)

• two key enzymes glycogen phosphorylase and glycogen synthase, are controlled by GPCR signaling

• Glycogen phosphorylase is activated in response to glucagon and epinepherine (low blood sugar; energy needed). Glucose is released from glycogen.

• GLycogen synthases is activated in response to insulin (elevated blood sugar). Glucose is polymerized into glycogen

A-Kinase-Associate Proteins (AKAPs)

Membrane anchored and have both PKA and PDE binding domains- tightly restricting where and how much cAMP is produced

Plasma membrane vs nuclear membrane AKAP localization in different cell types can control whether the primary response is enzymatic or transcriptional

Inhabitation of activated GPCRs

• active receptors only are phosphorylated by GPCR Kinases (GRKs) - this inhibits the ability of the receptor to engage G proteins- signaling is suppressed, a phenomenon called desensitization

• This then recruits proteins called β-arrestins, which bind the GPCR and enhance the block of transduction

• Ultimately β-arrestin recruits clathrin and promotes endocytosis of the activated receptor

• The receptor may be recycled back to the cell surface to once again receive signal or be directed to lysosomes for degradation making the cell unable to receive signal

• β-arrestin has also been shown to recruit a variety of intracellular signaling kinases (like SRC, shown here) that may activate additional transduction pathways from the membrane of endosomes before being degraded

G_⍺q IP₃ flowchart

ligand → GPCR → G_⍺q → Phospholipase C → + PIP₂ (phosphtidylinositol (4,5) bisphosphate) → IP₃ inositol (1,4,5) triphosphate: second messenger binds to IP₃ receptor on Er releasing Ca²⁺ → 1. Ca²⁺ regulates cell-type specific target proteins to generate appropriate response: usually indirectly via binding calmodulin or → DAG: diacyl glycerol not a second messenger, binds and activates some PKC proteins → PKC phosphorylates cell-type specific target proteins to generate appropriate response

G_⍺q DAG flowchart

ligand → GPCR → G_⍺q → Phospholipase C → + PIP₂ (phosphtidylinositol (4,5) bisphosphate) → DAG: diacyl glycerol not a second messenger, binds and activates some PKC proteins → PKC phosphorylates cell-type specific target proteins to generate appropriate response

G_⍺t flow chart

ligand → GPCR → G_⍺t (transduction) → phosphodiesterase (PDE) → +cGMP or cAMP depending on the PDE → GMP or AMP → cG/AMP signaling is halted

PHospholipase

One of serval enzymes that cleave various bonds in the hydropholic end of phospholipids

GPCRs signaling through the effector enzyme phospholipase C

G_⍺q - activates phospholipase C

• the G-protein effector enzyme phospholipase C cleaves PIP₂ into IP₃ (soluble and DAG (membrane bound)

• IP₃ opens ligand-gated CA²⁺ channels in the Er releasing Ca²⁺ into the cytosol which activates Protein Kinase C (PKC) and calmodulin

• DAG binds and cooperatively activates PKC

Phosphoinositides

A group of membrane-bound lipids containing phosphorylated inositol derivatives; some function as second messengers in serval signal transduction pathways

are phosphorylated forms of the lipid phosphatidylinositol

PI kinases

Phosphorylate specific -OH groups of inositol generating various PI forms - note difference in number and placement of phosphates

PI-4,5-bisphosphate [PI(4,5)P₂] (PIP₂)

in the case of GPCR signal transduction, the phosphinosotide PI-4,5-bisphosphate [PI(4,5)P₂] (PIP₂) is substrate for the effector enzyme phospholipase C itself activated downstream of certain GPCRs

G Protein-Coupled Receptors and Their Second Messengers: IP₃ and DAG

the effector enzyme phospholipase C is activated by a subset of heterotrimeric G proteins via either G_⍺0 or G_⍺q proteins

PLC cleaves PI(4,5)P₂ generating insoitol triphosphate (IP₃) and the membrane tethered diacylglycerol (DAG)

the sugar phosphate IP₃ is water soluble and can rapidly diffuse throughout the cell

IP₃ binds the IP₃ receptor on the SER surface. the receptor is a ligand-gated Ca²⁺ channel that once activated releases Ca²⁺ into the cytoplasm

depending on the cell type these ions bind and regulate diverse proteins that regulate different cellular activities

DAG remains membrane bound and allosterically activates the enzyme protein kinase C which phosphorylates serine and threonine residues on diverse target proteins

these proteins play varied roles in cell growth, differentiation, cell death, metabolism, and the immune response

PKC activation also requires Ca²⁺ release. only when bound to Ca²⁺ can PKC translocate to the membrane where it is activated by DAG

CaM Kinases

Ca²⁺ / calmodulin dependent protein kinases

important family of calmodulin targets

Depolarized State

When potential across the plasma membrane is less negative than that of the normal resting state of the cell

In the absence of light Rod cells are depolarized ~-30mV and secrete a neurotransmitter that stimulates neurons called bipolar cells

Rod cells

photoreceptors responsible for sensing light when it’s dark

Opsin

Once dark adapted, however, our eyes can begin picking up faint light through the action of GPCR opsin, located in flattened membrane disks in the outer segment of rod cells.

Rhodopsin

Composed of the GPCR opsin covalently attached to the light-absorbing pigment 11-cis-retinal

• when 11 cis-retinal absorbs a photon it rapidly isomerizes to the all-trans isomer

• this induces a conformational change in rhodopsin to the activated unstable intermediate meta-rhodopsin II

• meta-rhodopsin II binds and activates its cognate heterotrimeric G-protein generating G_⍺t-GTP (transducin)

• the all-trans-retinal moeity quickly dissociates from opsin (within milliseconds)

• It is converted by a series of enzymes back to the cis isomer and reattaches to opsin

Guanylyl Cyclase

In the depolarized state the enzyme guanylyl cyclase catalyzes production of high levels of the second messenger cGMP

Glutamate

neurotransmitter blocking neurotransmitter release by bipolar cells

Dark adapted rod cells and closing of cation channels

In the dark, our retinal are able to regenerate 11 cis-retinal. It can take up to 30 minutes for human eye to become fully dark adapted. One adapted however:

• Rhodopsin absorbs photons and is converted to meta-rhodopsin II

• meta-rhodopsin II functions as a GEF to catalyze Gαt exchange of GDP for GTP.

• Free GαtGTP binds and activates the enzyme phosphodiesterase (PDE)

• PDE hydrolyzes cGMP to GMP - dramatically lowering cGMP concentration

• Cation channels close and Na+ and Ca2+ influx ceases

• Membrane is transiently hyperpolarized.

Glutamate release is blocked.

Bipolar cells are no longer stimulated

They secrete their own neurotransmitters

Which are received by the ganglia that transmit the perception of light to the brain.

Rapid termination of rhodopsin signaling

1. signal termination: (~0.2 sec, essential for the temporal resolution of vision)

   • G_⍺T bound by a GAP complex (inset box)

   • Which stimulates rapid G_⍺t GTP hydrolysis to GDP

   • This reestablishes the inactive heterotrimeric G-protein

   • PDE is deactivated and cGMP accumulates- cation channels reopen

2. Also, the drop in Ca²⁺ activates guanylyl cyclase activating proteins. These are Ca²⁺ sensing proteins that are inactive when bound by Ca²⁺ .

3. Feedback repression of activated rhodopsin

   • Rhodopsin kinase- A GRK kinase that phosphorylate activated GPCRs Phosphorylation inactivates and desensitizes them to further stimulation

   • Arrestin proteins bind phosphorylated GPCRs preventing them from interacting with and activating G_⍺T - stimulation is transiently halted

   • phosphatases removes GPCR phosphorylation, arrestin is released and rhodopsin can again be excitied- there is no endocytosing of these GPCRs