Signal

Transduction – Lecture 11
1. Define the characteristics of receptor-ligand interactions
• Ligands – chemical messengers; generally involved in binding to receptors and initiation of chemical signaling pathway
o Nervous system –neurotransmitter neuropeptide
o Immune system – cytokines
o Endocrine – hormones (endocrine glands)
o Also includes retinoid, eicosanoids, growth factors, etc.
• Receptor – protein with specific ligand-binding region, capable of transmitting message; extracellular (polar) or
intracellular (nonpolar; hydrophobic)
• Second Messenger – non-protein molecules involved in signal propagation
o Includes cAMP, DAG (diacylglyceral) IP3 (inositol triphosphate)
• Signal Transduction – defined as conversion and propagation of specific information into cellular response
o High degree of specificity, often as a function of ligand binding site
o Non-covalent interactions – hydrogen bonding, electrostatics, hydrophobic interactions (VDW forces, etc)
o Limited expression
o High affinity of ligands for receptors, etc. (represented by dissociation constant, Kd)
o Typically involves amplification of signal – enzymes activating enzymes results in signal cascades

2. Explain the critical events in signal transduction processes
• Four Critical Events:
a) Specificity – signal molecule fits binding site on complimentary receptor
b) Amplification – when enzymes activate enzymes, number of molecules affected increases geometrically
c) Desensitization/Adaptation – receptor activation triggers feedback to inactivate/remove receptor
d) Integration –when two signals cause opposing processes, regulatory outcome results from integration of both

Quantifying Receptor-Ligand Binding
• Dissociation Constant (Kd) – quantifies receptor-ligand binding; indicates amount of ligand a receptor can detect

Ka = [RL]/[R][L] = k+1/k-1 = 1/Kd


Where R = receptor, L = ligand, RL = complex, K+1 = association
constant/rate of binding, K-1 = dissociation constant/rate of release

• Note that nonspecific binding accounts for ligands ‘sticking’ to other
proteins or neighboring molecules – not saturable
• Since Kd indicates relative detection of a ligand (by receptor), receptor
with weak binding (nM Kd) will be activated at much lower
concentration than receptor with mM Kd for same ligand
• Kd basically allows one to compare the relative binding capacity of
proteins/receptors to a particular ligand

Statchard Plot – allows for estimation of Kd and number of ligand binding sites (at equlibruim)

Bmax = Total number of binding sites = [R] + [RL]

[𝑹𝑳] 𝟏
= 𝑩 − [𝑹𝑳]
[𝑳] 𝑲𝒅 𝒎𝒂𝒙

Types of Signaling:
• Endocrine – signaling molecule transported via blood
• Paracrine – signaling molecule acts on neighboring cell
• Autocrine – signaling molecule acts on itself (secreting cell)
• Juxtacrine – molecule attached to signaling molecule acts on adjacent cell

3. Identify the major classes of receptors and be able to describe one example of each

Major Classes of Receptors:

G-Protein Coupled (GPCRs) – External [S] binds [R], activating GTP-G protein, which regulates [Enz] that generates X
• 7 transmembrane regions; intracellular trimeric G-protein (bound to GTP); produces cAMP (second messenger)
o Alpha, beta and gamma subunits; GTP forms a-GTP complex (GTPase activity)
• Multiple family members
o Gas – stimulatory G protein – stimulates cAMP production via adenylyl cyclase; bound GTP = active
o Gai – inhibitory G protein – inhibits cAMP production
o Inactivated via hydrolysis of GTP – determines length of activity (inactivation or regulatory action)
• Two main pathways: cAMP and phosphatidylinositol
• Adrenergic Receptor: four types – a1, a2, B1, B2
o Based on affinity/response to agonists (activating analogs) antagonists (inactivating analogs)
o Generally, adrenergic receptors bind catecholaminergic neurotransmitters (epi/norepi)
o Multiple conditions associated with adrenergic
receptors
§ Heart disease, migraines, anxiety, glaucoma

Beta Adrenergic Receptor (see right)
o Different functions in tissues/diseases due to
different adrenergic receptors and trimeric G-
proteins
o B2 in skeletal muscle – Epinephrine stimulates
cAMP production via Gsa-GTP binding to
o A2 in smooth muscle – Epinephrine inhibits
cAMP synthesis via Gai
o Glucagon regulates via signals GPCR in liver


• Termination of B-Adrenergic Signal:
o [Ligand] decreases below Kd
o Hydrolysis of GTP by Gas
o Conversion of cAMP to AMP via cyclic nucleotide phosphodiesterase
o Desensitization – occurs while ligand attached
§ BARK – Beta adrenergic protein kinase; recruited to membrane by Gsby,
where it phosphorylates Ser at C-terminus of receptors
Ø BArr (B-Arrestin) – binds to phosphorylated C-terminal domain of
receptor
Ø Receptor-BArr complex enters cell via endocytosis
§ Ultimately, arrestin dissociates, receptor dephosphorylated à returns
to cell surface
Phospholipace C (PLC) Pathway
• Uses Gaq G-protein; steps culminate in intracellular release of Ca2+, leading to
phosphorylation cascade via PKC
a) Hormone ligand binds to receptor – ligands include angiotensin II,
oxytonic, serotonin [5-HT1c]
b) Occupied receptor causes exhange of GDP for GTP on Gaq
c) Gaq-GTP complex activates PLC
d) PLCB binds PIP2, cleaves lipid to yield DAG and IP3
e) IP3 binds to endoplasmic reticulum receptor, releasing Ca2+
f) DAG and Ca2+ activate protein kinase C (PKC) at intracellular membrane
g) Phosphorylation of cellular proteins by PKC produces cellular responses to
hormone
• Activation of PKC achieved via Ser/Thr Kinase – serine/threonine specific PK
phosphorylations hydroxyl group of Ser or Thr
Calcium – Second Messenger
• Unstimulated cell [Ca2+] = 10-7M
• Calmodulin – enzyme contains 4 Ca2+ binding sites – EF hand motif
o Calmodulin activates Ca2+/calmodulin dependent protein kinases (CaM Kinases) and other enzymes
• Intracellular release of calcium is highly controlled, and Ca2+ release tends to exhibit oscillatory behavior

Cholera and Pertussis Toxins
§ Cholera – Vibrio cholera induces ADP-ribosylation of Arg in Gas subunit
Ø Inhibits GTPase activity, results in constitutionally active (perpetual) state
Ø Basically, toxin attaches ADP-ribose to Gas subunit, resulting in covalent activation of system
§ Pertussis – Bordetella pertussis induces ADP-ribosylation of Gai subunit [whooping cough]
Ø Modification prevents GDP-GTP exchange, and blocks inhibition of adenylyl cyclase
Ø Respiratory tract issues result from damage to ciliated epithelia, leading to vigorous coughing
Ø Basically, epithelia are unable to push debris-filled mucous from respiratory tract – coughing


Beta Blockers and Cardiovascular Disease
§ Beta Blockers slow heart rate and reduce force of contraction
§ Normally, activation of B-adrenergic receptors via epinephrine/norepinephrine activates adenylyl cyclase and PKA,
which phosphorylates cell survace calcium channels and proteins on sER
§ Phosphorylation leads to intracellular calcium release – calcium release from sER à muscle contraction
§ Beta blockers inhibit binding of epi/norepi to b-adrenergic receptor (B1 and B2) \reduce entry of Ca into myocytes
via inhibition of Ca release from sER
§ Common Treatment for…
Ø Hypertension
Ø Angina
Ø Arrhythmias
Ø Hypertrophy
Ø Heart Failure
Receptor Tyrosine Kinase (RTKs) – [S] binding activates tyrosine kinase activity by autophosphorylation
• Transmembrane proteins, with extracellular ligand binding domain
• Intrinsic kinase activity of cytoplasmic tail
• Dimerization of proteins facilitates autophosphorylation
• Important in the signaling of cellular growth – stimulates factors for transcription/translation
o VEGF – vascular endothelial growth factor
o PDGF – platelet derived growth factor
o EGF – epidermal growth factor
o NGF – nerve growth factor
o FGF – fibroblast growth factor
Insulin Receptor (INS-R)
o Two a- and two B- subunits – a binds insulin, B utilizes
ATP for kinase activity
o Autophosphorylation – 3 Tyr/B subunit; allows receptor
to phosphorylate othersà insulin receptor substrate 1
(IRS1)
a) When phosphorylated by INS-R, IRS1 activates PI-3K
b) PI-3K binds to p-IRs-1, converting PIP2 to PIP3
c) Akt (PKB) is phosphorylated by PDK1
Ø Akt phosphorylates glycogen synthase kinase 3
(GSK3), inactivating it and increasing glycogen
synthesis
Ø Akt stimulates incorporation of glucose transporter
(GLUT4) into membrane, increasing glucose uptake
d) PIP3 –specific phosphatase removes 3’ PO4
e) P-IRS-1 can also bind and inactivate PLCy à IP3 & DAG
o Note that phosphorylation of glycogen synthase kinase
(GSK3_ illustrates phosphorylation-induced inactivation
of an enzyme
§ Phosphorylation is generally associated with
activation, not inactivation
• Serine/Threonine Kinase Receptors
o Utilizes TGFB (transforming growth factor beta), which binds to TGFBR
§ TGFBR –dimer of type I and type II receptors
Ø 2 type I and 2 type II receptors; TGFB binds to type II receptor
Ø Activated type II receptor phosphorylates type I receptor à type I mediates downstream signaling
o Classic Smad Activation Pathway:
a) Type I receptor phosphorylates Smads
b) Activated Smads translocate to nucleus
c) Gene transcription is either promoted or inhibited, based on Smad involved

TGFB Signaling and Cancer:
§ Normally, TGFB functions as tumor suppressor – inhibits cell cycle progression, induces apotosis, or prevents
immortalization \ loss of function can be implicated in cancer formation
§ Tumor cells exhibit exaggerated TGFB secretion – autocrine and paracrine signals

• Cytoplasmic Tyrosine Kinases: Cytokines – small proteins that regulate behavior of cells (esp hematopoietic)
o Transmembrane receptor – no intrinsic signaling activity à binds cytoplasmic tyrosine kinases
o Ligand-induced Dimerization
a) Ligand binds to extracellular site (erythropoietin)
b) Receptor binds intracellular JAK (janus kinase); activated JAK phosphorylates receptor
c) Signal transducers and activators of transcription (STATs) bind to phosphorylated receptor
d) STAT phosphorylated by JAK to expose NLS (nuclear localization sequence)
e) Nuclear import/translocation and alterations to gene expression (transcriptional)

 Erythropoietin (Epo) – regulates RBC production; hypoxia induces production by kidneys
o Receptor found on common myeloid progenitor cells
o Common agent employed to treat anemia (Epogen/Procrit and Aranesp)
o Used in blood doping to improve O2 delivery

Polycythemia – Increase in RBCs associated with chronic oxygen deficiency
o Polycythemia vera caused by myeloproliferative neoplasm (erythropoietic stem cells)
o RBC count increases, blood thickens
o Caused by missense mutation (V216F) in JAK2
§ Creates constitutionally active species
§ Treatment with hydroxyurea or Ruxolitinib (Jakafi), a JAK inhibitor

Note that binding domains allow for varied signal transduction
• Conserved domains generally serve a functional purpose, such as binding to a ligand or mediation of enzymatic function
• Cytoplasmic kinases and adaptor proteins have many conserved binding domains
• Phosphates can serve as binding sites for PO4 on Ser, Thr, or Tyr
• Phosphatases remove PO4 residues, while kinases generally add them
• Sometimes, PO4 binding inhibits activity, such as GSK3 and Src

Receptor Guanylyl Cyclase – [S] binds to extracellular domain, stimulating formation of cGMP
• Enzymatic activity can be either transmembrane or cytosolic protein
• RGC converts GTP to cGMP (similar to cAMP)
• Ligand – atrial natriuretic factor (ANF) myocytes produce when blood volume increases cardiac load
• Tissue-dependent message-- Kidney/Intestinal ion transport and water retention
o Intestines – increases excretion of Cl-; activated by guanylin, a common target of bacterial endotoxins
§ Increased Cl- leads to increased H2O excretion
o Kidney – increases excretion of Na+; activated by ANF
§ Increased Na+ leads to increased H2O excretion
o Smooth Muscle – role in relaxation
§ Nitric Oxide (NO) enters cell (diffusion), binding to guanylyl cyclase to activate cGMP production
§ Elevated cGMP causes decrease in intracellular Ca2+, since calcium is critical for contractility

Viagra and Cytosolic Guanylyl Cyclase:
o Elevated [cGMP] leads to vasodilation
o cGMP phosphodiesterase (PDE_ enzymatically converts cGMP to GMP, inactivating guanylyl cyclase signals
o Viagra blocks PDE5 activity \ maintains elevated [cGMP] to relax smooth muscle of vessels, leading to vasodilation


Adhesion (Integrin) – Binds molecules in ECM, changes conformation \ alters interaction with cytoskeleton
• Cell-cell and cell-ECM interactions via integrins
• Involved in development, migration, adhesion, and tumor growth/metastasis
• Heterodimeric 18 a and 8 B chains; 24 different integrins
o Ligand binding requires divalent cations (Mg2+ or Mn2+ -- cofactors)
o Ca2+ in a-chain for stabilization
• Expression is variable – most cells express more than one type
• Chains are able to interchangeably associate – modulates specificity
• Attachment to extracellular matrix involves other proteins
o Collagen, laminin, fibronectin (Fn) and fibrinogen (Fbrn)
o Mediated by RGD (Fn, Fbrn)
• Bidirectional Signal Transmission:
o “Outside-in” vs “inside-out” – signal may originate intracellular or extracellularly
o Integrins aren’t always “on” – may require activation
o Conformational changes via ligand binding induce activation
o No intrinsic signaling activity on short, cytoplasmic tails

 Integrins and Clotting:
o Reduced Clotting – Glanzmann thrombasthenia
§ Autosomal recessive; often in population with marriage among close relatives
§ Mutation in a or B subunit of aIIbB3 on platelets – activated in clotting cascade
Ø Excessive bleeding occurs with minor injury
§ Symptoms: mucosal, givgival, GI bleading
§ Diagnosis involves complete blood cell count, measurement of clotting time
o Excessive Clotting – results from impaired regulation of platelet adhesion
§ Pathological clot formation may lead to stroke or heart attack
§ Treatment involves blocking extracellular ligand binding
§ Antiplatelet drugs (glycoprotein IIb/IIIa inhibitors) include Tirofiban (Aggrestar) and Eptifibatide (Integrillin)

Gated Ion Channel – Open or close in response to concentration of [S] or membrane potential (∆Vm)
• May be triggered by either ligand binding (hormones/NTs) or change in Vm via action potential, etc.
• Transports Na, K, Ca or Cl ions (cationic and anionic) – huge role in neurotransmission and muscle cells
• Intracellular environment negative relative to extracellular – Vm typically -50mV to -70mV
• Opening/closing of channels causes diffusion of ions across electrochemical gradient, resulting in either depolarization
(Na+ channels; Vm increases) or hyperpolarization (K+ channels; Vm decreases below resting potential)
o Electrical gradient refers to the relative charge across membrane; influenced by proteins
o Chemical gradient refers to the relative concentration of solutes in solution
o Gradient is actively maintained via active transport (NaK-ATPase activity)
o Ions stop diffusing through channel when equilibrium potential is achieved (Na = +70mV; K = -98mV)
• Na channels open in response to depolarization, moving Na+ into cell (negative Vm, low [Na+]in)
o Note refractory period associated with Na channels is important for unidirectional propagation in axons
• K channels open in response to Na+ influx/depolarization; K+ moves out of cell (high [K+]in)
• Calcium channels open in response to changes in Na+ and K+; calcium moves into cell (low [Ca]in)
o Calcium flow can trigger neurotransmitter release at synaptic cleft
• Ligand Gated Ion Channels – Nicotinic AChR
o Primary conduction mechanism between neuron and muscle cell, ant NMJ
o Ach binds to binding sites, causing conformational change in M2 helices to shift
§ Closed state – bulky hydrophobic Leu sidechains block channel
§ Open state – hydrophobic groups shift; polar residues line channel and allow passage of ions
+ 2+ +
Ø Na tends to be fastest; Ca and K may also enter channels.
o Termination – involves diffusion/reuptake of ACh and/or degradation via acetylcholinesterase

Nuclear Receptor – Steroid binding allows specific
modulation of gene expression via receptor
• Hormones can be extracellular, cytosolic or nuclear
• Water soluble and amine hormones tend to act
extracellularly (insulin, epinephrine) -- RAPID
• Hydrophobic (steroid, retinoid, thyroid) hormones
tend to act in the nucleus
o Delayed action – alter protein concentrations via
changes in gene expression (transcriptional)
o Hydrophobic hormones may readily diffuse
through membrane
o May act on either cytoplasmic or nuclear
receptors
• HRE (Hormone Response Elements) – signal related;
modify rate of transcription for specific genes
• Hormone receptors maintain conserved DNA binding
domains w/ 2 zinc fingers (nucleotide recognition)
o Ligand-binding domain on C-terminus; variable
• Transcriptional activation occurs via interaction with
various transcription factors
Clinical Targets of Hormone Signaling:
1) Tamoxifen – estrogen receptor antagonist; effective on estrogen sensitive cancers
2) Mifeprosteone (RU486) – progesterone analog; blocks action of natural ligand (competitive inhibition); necessary for
implantation after fertilization

4. Understand how changes in signal transduction pathways can lead to disease

See examples above in blue boxes for specifics. Generally speaking, signal transduction is a necessary aspect of coordinating
cellular processes, particularly in the context of development, gene regulation, and regular cellular function.

Disruption of any of these processes via competitive inhibition, genetic error/mutation, or permanent/covalent modification
may result in substantial pathology, especially when related to protein synthesis

5. Recognize how signal transduction pathways can be targeted for therapeutic intervention.

Although signal cascades may be a prime target for pathology and disease, the same mechanisms can be utilized to
disruptively target the intracellular mechanism of pathogenic organisms.

The variable and manipulable nature of second messenger systems makes prime targets for therapeutic intervention,
especially since the ligands for many biological receptors can be selectively synthesized/emulated, either directly or via
analogs/agonists/antagonists, etc.