BIOL 4150: Cellular Regulation

Lecture # 2

Mahmoud Sirdah, PhD

Professor of blood pathophysiology and genetics
Life science Building, 102G
Email: msirdah@[Link]
 Preprogrammed Responses
• A Cell carries out many functions at any time.

• However, some cellular functions or processes remain inactive until a triggering

signal is received

• Upon Ligand-receptor binding, a preprogrammed sequence of signal transduction

events are initiated inside the cell.

• Signal-induced gene expression may lead cell to express new receptors that make
it sensitive to a new set of signals for future functions.
 Quantitative ligands-receptors interactions: receptor saturation

• Ligand -receptor binding resembles an

enzyme-substate interaction in terms of
receptor saturation or occupied receptor.

• At low ligand concentration, receptor

occupation is proportional to the
concentration of free ligand in solution.

• At high ligand concentration more and

more receptors become occupied, until
most receptors are occupied—a
condition known as saturation.

More increases in ligand concentration will not affect the target cell further.
Qualitative ligands-receptors interactions: receptor affinity
• Receptor affinity >>> the qualitative relationship between ligand
concentration and receptor saturation

• Receptor-ligand high affinity >>> almost all receptors are occupied

at low concentrations of its ligand

• Receptor-ligand low affinity >>> high concentration of ligand to

occupy most receptors.
 Receptor affinity
• Receptor affinity is described quantitatively in terms of the dissociation constant (KD)

• Dissociation constant (KD) >>> concentration of free ligand at which 50 % the receptors
are occupied.
• Two ligands with different receptor affinities : Ligand 1 and Ligand 2

• KD2 < KD1 >>>>>> Ligand 2 has higher affinity to the receptor
 Co-receptors
• Ligand-receptor interaction can be affected or influenced by co-receptors on the cell
surface.
• Co-receptors help or facilitate primary receptor-ligand interaction through their
physical interaction with the primary receptor.

• Co-receptors provide additional regulation level of receptor-ligand interaction.

• Co-receptors, also known as accessory receptors

• Co-receptors are found on the cell surface and binds signaling molecule (ligand) in
addition to a primary receptor.

• Co-receptors facilitate ligand recognition and initiation of signal transduction

processes.
 The T-cell Co-receptor CD4
• Co-receptors strengthen T-cell responses by many orders of magnitude >>>> beyond a million-
fold !!!!.
• In one model (The Lck Recruitment Co-receptor Model) :
• T-cell receptor (TCR) triggering is initiated by TCR- pMHC binding (no signaling yet).
• CD4 is T-Cell co-receptor that binds to the same pMHC ligand
• CD4 is directly associate with the TCR-phosphorylating kinase (Lck) that is responsible for
phosphorylating the T cell receptor (TCR) at the start of T cell signaling
 The Lck Recruitment Co-receptor Model in TCR Signaling

• Initiation of TCR signaling requires the phosphorylation of TCR intracellular tails.

• Ligand-bound TCR alone is not able to transduce the activation signal (phosphorylation)
to inside the T-Cell.

• Co-receptors (e.g. CD4) play distinctive function in bringing (Lymphocyte-specific

protein tyrosine kinase) Lck to the ligand-bound TCR complex >>>>> thus
phosphorylating TCR intracellular tails >>>> activation of TCR and signal transduction
Models of Co-receptor Contributions to TCR Signaling
 Signal Amplification
• Signal amplification is an important feature in the
signal transduction pathways.

• Very small quantities of a ligand are usually enough

to trigger a response from a target cell.

• Strong response of the target cell usually results from

a signaling amplification cascade with the responding
cell.

• Through the cascade, signaling intermediates

stimulate the production of many molecules required
for the following step.

• This amplifies the effects of a single receptor-ligand

binding.

• e.g. breakdown of glycogen in liver in response to

epinephrine.
• A single epinephrine molecule can stimulate the
release of about 108 of glucose molecules from
glycogen.
 Signal Transduction Pathways
• Cells use a limited number of basic signaling pathways:

• Ligand-gated ion channels

• G protein–coupled receptors
•
• Enzyme-coupled receptors

• Nuclear receptors
 Ligand-gated ion channels (LGICs)

• LGICs are transmembrane receptor proteins (extracellular, intracellular

and transmembrane domains).

• LGICs contain a pore for regulated flow of selected ions.

• Ion influx is passive and driven by the electrochemical gradient of the

ion.

• Channels are opened, or gated, by ligand binding >>>> triggering

conformational changes >>>> ion influx >>> signal transduction.

• e.g. Nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid

(GABA) receptors, and Glycine receptors
Structure of pentameric ligand-gated ion channels
 G protein–coupled receptors (GPCRs)
• The largest and most diverse group of membrane receptors in eukaryotes.
• Bind a great variety of signaling molecules or ligands
• Share a common architecture.
• Cell surface transmembrane receptors
• Seven segments of a single polypeptide that pass through and span the entire width
membrane seven times
• Also known as seven-transmembrane receptors
• Rely on GTP binding
• Signaling molecules (Ligands) bind to pockets of the extracellular loops
 Enzyme-coupled receptors (e.g protein kinases)
• Receptors that are coupled with and activate cytosolic enzymes.
• Upon binding to their ligand, the receptor kinase activity is stimulated, and they
transmit signals through a cascade of phosphorylation events within the cell.
• e.g. Receptor Tyrosine Kinases, Receptor Serine-Threonine Kinases
 Nuclear receptors
• Nuclear receptors are a family of ligand-activated transcription
factors.
• Hydrophobic ligands can act on receptors in the cytosol
• Receptor-ligand binding>>>> translocate (receptor-ligand
complex) into the nucleus and regulate gene transcription.
• Hydrophobic messengers or ligands that bind to intracellular
receptors are:
• Steroid hormones derived from cholesterol
• Retinoids, derived from vitamin A.
 G Protein–Coupled Receptors
• Naming?
• Ligand binding causes a change in receptor conformation that activates a particular guanine-
nucleotide binding protein (G protein) in the cytoplasmic side of the cell membrane.
• G Protein–Coupled Receptors act via hydrolysis of GTP

Part of the activated G protein binds to a target protein (e.g. an enzyme or a channel
protein) and altering their activity (active inactive).
The Structure of GPCRs

• All GPCRs have a similar structure regardless of • GPCR forms seven transmembrane α
amino acid sequence differences. helices connected by alternating
cytosolic or extracellular loops.
• GPCR is a single polypeptide chain
• messenger-binding site on the
• N-terminus is exposed to the extracellular fluid extracellular portion.
• C-terminus is in the cytosol • Cytosolic loops allow the receptor to
interact with only certain types of G
proteins.
 G protein structure
• Two different classes of G proteins:
• The large heterotrimeric G proteins and associated with GPCRs and mediating signal
transduction.
• The small monomeric G proteins, not associated with GPCRs
• They include RAS superfamily proteins
• known as small GTPases
• Play essential roles in cell differentiation and proliferation
 GPCR associated G protein structure
• Heterotrimeric and is made up of three different subunits: α, β and γ.
• Gα subunit is the largest, binds to GDP or GTP.
• Use GDP to control their signaling cycle
• GDP is bound to α-subunit >>> inactive G protein.
• Replacement of GDP with GTP >>>> detaches G-protein α-subunit >>> activates G
protein >>>> deliver its signal
• Gβ and Gγ subunits form a stable dimeric complex >>>> can initiate other transduction
pathways.
 Classes of G-proteins
• Four classes of G-protein based on their α subunits (:
• Each α subunit stimulates an enzyme >>> either increase or decrease second messenger levels
• Stimulators of signal transduction>>>> Gαs >> increase cAMP levels (activates adenylyl
cyclase activity pathway)
• Gαq >>> Activates inositol phosphate pathway (activates phospholipase Cβ)
• Inhibitors of signal transduction >>> Gαi>> Inhibits cAMP pathway (inhibits adenylyl cyclase
activity).
• G α12/13 activate small GTPase families (Rho family GTPase signaling) controlling cell
cytoskeleton remodeling, and thus in regulating cell migration.
 GPCR activation
• Ligand -GPCR binding >>> Receptor conformational changes >>> causes receptor binds a
G protein (GαGDPβ)
• Binding of G protein (GαGDPβ) to GPCR >>>> releases GDP from the Gα subunit >>>>
acquiring GTP >>> detachment of the Gα subunit >>>> G β subunits keep associated with
the receptor or initiate signal transduction events
• According to G protein type>>> free GTP-Gα subunit and/ or the Gβ complex >>> initiate
signal transduction events
 G protein classical pathway

Exchange of GDP for GTP in Gα subunit >>>> dissociation of Gα & Gβ

subunits>>> interaction with downstream effectors (e.g. adenylyl cyclase and Gβγ
activation of ion channels) .
 GPCR activation
• GTP-Gα subunit or the Gβ complex >>>> bind to enzymes / proteins >>> initiate signal
transduction events .
• Persistence of G protein activity only if:
• Gα is bound to GTP
• Gα and Gβ subunits remain dissociated

• If Gα hydrolyzes its GTP (GDP + Pi) >>>> it reassociates with Gβ>>>> inactive G
protein >> shut down of signal transduction pathway.

• Some Gα proteins are not efficient in GTP hydrolysis >>> regulators of G protein
signaling (RGS) proteins >>> bind to Gα >>> stimulate GTP hydrolysis.

• (RGS) proteins are GTPase activating proteins (GAPs) >>> very important regulators of
G protein function.
 GPCR regulation via: 1. G protein– coupled receptor kinases

• GPCRs regulation via:

• Phosphorylation of GPCR cytosolic domain a.a>>> desensitization of GPCR.
• Phosphorylation of GPCR by:

• G protein– coupled receptor kinases (GRKs) >> act on activated receptors >>> heavily
phosphorylate GPCR cytosolic domain>>> β-arrestin binding >>>> inhibition of GPCR
ability to associate with G proteins.
 GPCR regulation via:2. Protein kinase A
• Phosphorylation of GPCR cytosolic domain a.a>>>
desensitization of GPCR. (via GRKs)

• Protein kinase A (PKA), which is itself activated by G

protein–mediated signaling, can also phosphorylate

other amino acids on the receptor. (negative feedback

during cell signaling

• Ligand binding >>> activates Gαs protein >>> increase adenylate cyclase signaling >>
cAMP and PKA activity.

• PKA phosphorylates GPCR >>> alters the receptor coupling from Gαs to Gαi (G protein
switching).
• Activated Gαi yields a decrease in cAMP production and decrease PKA activity>>
shutdown of signal transduction .
• (GPCRs –Ligands binding >>> G protein heterotrimers dissociate>>> active Gα and Gβγ
subunits.
• Four major classes of Gα subunits >>> each controls different downstream signal transduction.
• Gαs activates adenylyl cyclase >>>> stimulates cAMP production.
• Gαi inhibits adenylyl cyclase >>>>> inhibits cAMP production.
• Gαq activates phospholipase Cβ (PLCβ) >>>>> produces 2nd messengers' inositol trisphosphate
(IP3) and diacylglycerol (DAG)
• Gα12/13 >>>>> activate small GTPase families .
• Gβγ can also affect downstream signaling by binding to scaffold proteins or biding to
membrane ion channels .

Biding of acetylcholine (Ligand) to its G protein–coupled receptor >>> activates the

Gβcomplex >>> regulates K+ channels opening.
 Second Messengers Production Is Regulated by Some G Proteins
• G protein–mediated signaling events involve the
formation or release of second messengers (e.g.
cAMP and Ca2+ ions).
• Adenylyl cyclase produce cAMP from cytosolic
ATP
• Adenylyl cyclase is an inactive membrane- bound
enzyme >>> binding to activated Gαs subunit >>>
actives adenylyl cyclase >>>> converts ATP to
cAMP.
• Ligand leaves the receptor >>> activation ends
>>>> GTP is hydrolyzed to GDP>>> Gαs
dissociates from adenylyl cyclase >> inactive.
• cAMP molecules in the cytosol are hydrolyzed to
AMP by the phosphodiesterase.
• Once Gαs subunit hydrolyzes its bound GTP to
GDP >>>> inactive state >>> No more production
of cAMP>>> the Gαs reassociates with the Gβss
complex.
• phosphodiesterase degrades cAMP >>>>
shutdown of the signal transduction pathway.
Adrenaline binding >>>initiates signal transduction cascade of reactions inside the cell.
1- activations of adenylyl cyclase
2- cAMP production
3- cAMP activates protein kinases (PKA)
4- PKA enter the nucleus and affect transcription of target gene.
 cAMP targeting protein kinase A (PKA)

• Protein kinase A (PKA) is the main intracellular target

for cAMP

• PKA phosphorylates a wide variety of cellular proteins

• cAMP regulates the activity of PKA by causing the

detachment of its two regulatory subunits from its two

catalytic subunits.
 The activation and inhibition of cAMP-dependent protein kinase A (PKA)

cAMP absent
• PKA is an inactive tetrameric holoenzyme
• two regulatory (R)
• two catalytic subunits (C).

cAMP present
• 2 cAMP molecules binds to each R subunit at
allosteric sites
• Conformational changes
• Decrease affinity between the R and the C
subunits
• Separation of PKA holoenzyme into a
regulatory subunit dimer and two monomeric
catalytic active subunits.

• Active PKA phosphorylates their substrates >>> leading to further downstream signaling
 Some effects of increased cAMP
• An increase in cAMP concentration can produce different effects (preprogrammed
response) in different cells.
• In skeletal muscle and liver cells>>> stimulates glycogen breakdown
• In cardiac muscle>>> strengthens heart contraction
• In smooth muscle >>>>> inhibits muscle contraction
• In blood platelets >>>> inhibits platelets mobilization during blood clotting
• In intestinal epithelial cells >>> secretion of salts and water into the lumen of the gut.

• Artificially, cAMP concentration can be increased by:

• Stimulating cAMP production directly
• Theophylline is used to treat asthma because it relaxes bronchial smooth muscle.

• Inhibiting the enzyme phosphodiesterase that degrades cAMP.

• Methylxanthines (caffeine and theophylline) are phosphodiesterase inhibitors