Transcript Hormone Receptors on the Plasma Membrane

Hormone Receptors on the
Plasma Membrane
Characteristics of Receptors in General
Five Groups of Membrane-Bound Receptors
The G Protein-Coupled Receptor Superfamily
Signal Transduction through Cyclic AMP
Signal Transduction through Phospholipase C
Role of Calcium
Role of Protein Kinase C
General Characteristics of Receptors
• Receptors bind hormones, resulting in a biological
response
• All receptors exhibit general characteristics:
- Specific Binding (structural and steric
specificity)
- High Affinity (at physiological concentrations)
- Saturation (limited, finite # of binding sites)
- Signal Transduction (early chem event must
occur)
- Cell Specificity (in accordance with target organ
specificity).
Specific Binding
• A receptor will only bind (recognize) a certain
hormone, or closely related hormones.
LH
LH
hCG
hCG
LH Receptors
FSH
FSH
Receptors Have High Affinity
• In the bloodstream, there are thousands of different
peptides.
• Hormones are present in very small quantities
(nanogram or picogram).
• Receptors must therefore be very sensitive to the
presence of a hormone (they must be able to bind the
hormone even if it is present in low amounts).
• Thus, they have high affinity (ability to bind at low
hormone concentrations).
Analysis of Receptor Binding Sites
TESTIS
125
Na- I + hCG
lactoperoxidase
method
I 125 -hCG
TRACER
Leydig/Interstitial
Cells
RT O/N
WASH, PBS
Centrifuge
Count Pellet
CPM
Seminiferous
Tubules
Criteria for hormone-mediated events
• Receptor must possess structural and steric specificity for a
hormone and for its close analogs as well.
• Receptors are saturable and limited (i.e. there is a finite number of
binding sites).
• Hormone-receptor binding is cell specific in accordance with target
organ specificity.
• Receptor must possess a high affinity for the hormone at
physiological concentrations.
•Once a hormone binds to the receptor, some recognizable early
chemical event must occur.
• Affinity: The tenacity by which a drug binds to its receptor.
– Discussion: a very lipid soluble drug may have irreversible effects;
is this high-affinity or merely a non-specific effect?
• Intrinsic activity: Relative maximal effect of a drug in a particular
tissue preparation when compared to the natural, endogenous ligand.
– Full agonist – IA = 1 (*equal to the endogenous ligand)
– Antagonist – IA = 0
– Partial agonist – IA = 0~1 (*produces less than the maximal
response, but with maximal binding to receptors.)
• Intrinsic efficacy: a drugs ability to bind a receptor and elicit a
functional response
– A measure of the formation of a drug-receptor complex.
• Potency: ability of a drug to cause a measured functional change.
Receptors have two major properties: Recognition and Transduction
Recognition: The receptor protein must exist in a conformational state that allows for
recognition and binding of a compound and must satisfy the following criteria:
•Saturability – receptors exists in finite numbers.
•Reversibility – binding must occur non-covalently due to weak intermolecular forces
(H-bonding, van der Waal forces).
•Stereoselectivity – receptors should recognize only one of the naturally occurring
optical isomers (+ or -, d or l, or S or R).
•Agonist specificity – structurally related drugs should bind well, while physically
dissimilar compounds should bind poorly.
•Tissue specificity – binding should occur in tissues known to be sensitive to the
endogenous ligand. Binding should occur at physiologically relevant concentrations.
The failure of a drug to satisfy any of
these conditions indicates nonspecific binding to proteins or
phospholipids in places like blood or
plasma membrane components.
Receptors have two major properties: Recognition and Transduction
Transduction: The second property of a receptor is that the binding of an
agonist must be transduced into some kind of functional response
(biological or physiological).
Different receptor types are linked to effector systems either directly or
through simple or more-complex intermediate signal amplification
systems. Some examples are:
• Ligand-gated ion channels – nicotinic Ach receptors
• Single-transmembrane receptors – RTKs like insulin or EGF receptors
• 7-transmembrane GPCRs – opioid receptors
• Soluble steroid hormones – estrogen receptor
Predicting whether a drug will cause a response in a
particular tissue
Factors involving the equilibrium of a drug at a receptor.
• Limited diffusion
• Metabolism
• Entrapment in proteins, fat, or blood.
Response depends of what the receptor is connected to.
• Effector type
• Need for any allosteric co-factors – THB on tyrosine hydroxylase.
• Direct receptor modification – phosphorylation
Receptor theory and receptor binding.
Must obey the Law of Mass Action and follow basic laws
of thermodynamics.
• Primary assumption – a single ligand is binding to a
homogeneous population of receptors
NH+3
COO-
kon/k1
[ligand  receptor]
[ligand] + [receptor]
koff/k2
• kon = # of binding events/time (Rate of association) =
[ligand]  [receptor] kon = M-1 min-1
• koff = # of dissociation events/time (Rate of dissociation) =
[ligand  receptor] koff = min-1
• Binding occurs when ligand and receptor collide with the
proper orientation and energy.
• Interaction is reversible.
• Rate of formation [L] + [R] or dissociation [LR] depends
solely on the number of receptors, the concentration of
ligand, and the rate constants kon and koff.
•At equilibrium, the rate of formation equals that of dissociation so
that:
[L]  [R] kon = [LR] koff
KD = k2/k1 = [L][R]
[LR]
*this ratio is the equilibrium dissociation constant or KD.
KD is expressed in molar units (M/L) and expresses the affinity of
a drug for a particular receptor.
• KD is an inverse measure of receptor affinity.
• KD = [L] which produces 50% receptor occupancy
• Once bound, ligand and receptor remain bound
for a random time interval.
• The probability of dissociation is the same at any
point after association.
• Once dissociated, ligand and receptor should be
unchanged.
• If either is physically modified, the law of mass
action does not apply (receptor phosphorylation)
• Ligands should be recyclable.
Receptor occupancy, activation of target cell
responses, kinetics of binding
•Activation of membrane receptors and
target cell responses is proportional to the
degree of receptor occupancy.
•However, the hormone concentration at
which half of the receptors is occupied by a
ligand (Kd) is often lower than the
concentration required to elicit a halfmaximal biological response (ED50)
Receptor Fractional Occupancy
[LR]____ = [LR]___
[Total Receptor] [Rf] + [LR]
[R] = KD • [LR]
[L]
*now substitute the KD equation.
 F.O. = [Ligand]
[Ligand] + KD
Use the following numbers:
[L] = KD= 50% F.O.
[L] = 0.5 KD = 30% F.O.
[L] = 10x KD = 90%+ F.O.
[L] = 0= 0% F.O.
Fractional Occupancy
F.O. =
100
50
0
Ligand Concentration
Assumptions of the law of mass action.
• All receptors are equally accessible to ligand.
• No partial binding occurs; receptors are either
free of ligand or bound with ligand.
• Ligand is nor altered by binding
• Binding is reversible
• Different affinity states?????
Studies of receptor number and function
•
•
We can directly measure the number (or density) of receptors in the LR complex.
Ligand is radiolabeled (125I, 35S. or 3H). Selection of proper radioligand:
– Agonist vs. antagonist (sodium insensitive)
– Higher affinity for antagonists
– Longer to steady state binding
•
•
•
Saturation binding curve-occurs at steady state conditions (equilibrium is
theoretical only).
Demonstrates the importance of saturability for any selective ligand.
Provides information on receptor density and ligand affinity and selectivity.
Scatchard transformation
• Y-axis is Bound/Free (total radioligand-bound)
• X-axis Bound (pmol/mg protein)
• Straight lines are easier to interpret.
• The amount of drug bound at any time is solely
determined by:
– the number of receptors
– the concentration of ligand added
– the affinity of the drug for its receptor.
• Binding of drug to receptor is essentially the same as
drug to enzyme as defined by the Michelis-Menten
Equation.
Thus, to reiterate…,Calculating Affinity
% binding
• Take a cell which has the receptor on it (ie, granulosa cells
with FSH receptor).
• Prepare membrane homogenate.
• Incubate membranes with increasing amounts of labeled
hormone.
• Determine how much binding of hormone occurs at each
dose.
• Dissociation constant (Kd) is dose where 50% of maximal
binding occurs.
100
50
Kd
0
10
30
100
Dose of Hormone
300
1000
Thus, to reiterate…,Saturation
% binding
• There is a finite limit to the numbers of receptors
which can be on a cell.
• Therefore, there’s a maximum amount of binding
which can occur (all receptors are saturated)
100
saturation
50
0
10
30
100
Dose of Hormone
300
1000
Biological Response to Ligand Binding
• A receptor not only binds hormone; there must
also be a biological response from the cell (e.g.,
increased transcription, phosphorylation, etc.)
• This is also called “signal transduction”.
• The biological response can result from:
- the ligand itself (e.g., Fe, LDL)
- the receptor (e.g., increased cyclic AMP,
transcription, phosphorylation)
Determinants of Biological Response
• The strength of the response of the cell to the
hormone depends upon three factors:
1) the amount of hormone present to bind to the
receptors
2) the numbers of receptors on the cell
3) the affinity of the receptor for the hormone
(how much hormone do you need to get receptor
binding?)
Regulation of Receptor Number:
the Phenomenon of Spare Receptors
• We know that cells typically have about 20 times more
receptors than is needed for a maximal biological response.
• A complete biological response occurs after binding to only
5% of the receptors on a cell.
• This remaining 95% are called “spare receptors”.
• Why have spare receptors?
100
Biological
Response
(% max)
50
0
0
25
50
75
Receptor Occupancy (%)
100
Effect of Decreasing Receptor Number in a
Cell Which Does Not Have Spare Receptors
% of receptors occupied
• No change in affinity.
• Decrease in maximal biological response.
100
100
75
75
50
% maximal
50 response
25
25
Kd
0
10-11
10-10
0
10-9
Hormone Concentration (M)
Effect of Decreasing Receptor Number in a
Cell Which Has Spare Receptors
• In this example, assume you need 5000 receptors occupied for
maximal biological response.
• If you start w/ 20,000 receptors occupied, decreasing receptor
number does not change receptor affinity (Kd).
0
20,000
15,000
Number of
receptors bound 10,000
50
5,000
75
# Receptors
for maximal
biological
response
0
Kd
Hormone Concentration (M)
Effect of Decreasing Receptor Number in a
Cell Which Has Spare Receptors
• No change in maximal biological response (unless you go
below 5000 receptors/cell).
• Requires higher dose of hormone to obtain maximal
response.
100
10,000
5000
20,000
R/cell
80
% Maximal
Biological
Response
60
2500
40
20
0
10-11
10-10
10-9
10-8
Hormone Concentration (M)
10-7
Hormones, Agonists, and Antagonists
• Substances other than a receptors normal hormone
may exist (or be made).
• Each substance that binds to a receptor has an
intrinsic activity (related to the resulting biological
response).
Substance
hormone
superagonist
partial agonist
antagonist
Intrinsic Activity
100% (by definition)
>100%
<100%
0%
Antagonists
• If a substance binds to a receptor but does not cause
a biological response, it blocks the natural hormone
from binding to it.
• Example: RU486 binds to the progesterone receptor,
but does not cause a response.
progesterone
RU486
hormone binding
domain
transcriptional
domain
DNA
PR
Regulation of Biological Response at the
Receptor Level
• The strength of signal transduction can be regulated at the level
of the receptor by several mechanisms:
1) change the affinity of the receptor (make it bind more difficult
or easier to bind hormone). This usually doesn’t happen.
2) change the numbers of receptors on the cell (common)
- internalization and degradation of receptors
- occupancy of receptors (prevents hormone binding)
- gene expression/synthesis
3) change the signal transducing ability of the receptor (usually
for rapid regulation)
- phosphorylation (usually inhibits receptor activity)
- G protein uncoupling (stay tuned)
Plasma Membrane-Bound Receptors
• Recall that peptide hormones are polar, and
cannot readily cross the cell membrane.
• Therefore, their receptors must be on the outside
surface of the cell.
Types of Plasma Membrane Receptors
There are five basic types of membrane bound receptors
(grouped by signal transduction method):
• tyrosine kinase receptors
• receptors that are closely linked to tyrosine kinases
• receptors with guanylyl cyclase activity
• receptors that serve as transporters
• G protein-coupled receptors
Signal Transduction by
Plasma Membrane Receptors
1) Receptors with intrinsic tyrosine kinase activity. Binding
of hormone to the receptor induces the phosphorylating
activity of the receptor.
Example: Insulin receptor
extracellular domains
(ligand binding)
plasma membrane
tyrosine phosphorylase
domains
phosphorylated enzyme (altered activity)
Signal Transduction by
Plasma Membrane Receptors
2) Receptors that are closely linked to tyrosine kinases.
These activate cytoplasmic tyrosine kinase enzymes.
Example: Growth Hormone Receptor
associated
tyrosine
kinase
phosphorylated enzyme
Signal Transduction by
Plasma Membrane Receptors
3) Receptors with Guanylyl Cyclase Activity. Binding to the
receptor activates guanylate cyclase region of the
receptor, causing conversion of GTP to cyclic GMP.
Example: Atrial Natriuretic Peptide Receptor
GTP
guanylate
cyclase
cyclic GMP
ion channels
protein kinase G
phosphodiesterase levels
Signal Transduction by
Plasma Membrane Receptors
4) Receptors that serve as transporters. These move the
ligand inside the cell, where they have an effect. (Not
typical for hormones).
Example: Iron, transported by transferrin receptor
iron
transferrin
Signal Transduction by
Plasma Membrane Receptors
5) G Protein-coupled receptors. (The largest group!)
These receptors are coupled with guanine nucleotidebinding proteins (G proteins), which activate various
signaling pathways.
Examples: Receptors for LH, FSH, TSH, GnRH, dopamine,
serotonin, glutamine, parathyroid hormone, interleukins,
etc.
G Protein-Coupled Receptor Superfamily
• Common structural features:
- an amino terminus hormone-binding domain
- seven hydrophobic transmembrane domains
- a carboxyl terminus, intracellular domain
-NH2
COOH-
G Protein-Coupled Receptor Superfamily
• Common functional features:
- binding to the receptor activates a G protein
- each receptor is associated with a specific type of G
protein
- each G protein type has different functions:
- Gs: stimulates cyclic AMP
- Gi: inhibits cyclic AMP
- Go activates phospholipase C
How G Proteins Work
• G proteins are composed of three subunits: alpha (a),
beta (b), and gamma (g).
• In the inactive state, the three subunits are associated
with the receptor at the plasma membrane. The alpha
subunit has a guanosine diphosphate attached.
NH2
g
COOH
a
b
GDP
How G Proteins Work (cont.)
• When hormone binds, the GDP leaves the alpha
subunit, and is replaced by a GTP.
• The alpha subunit then goes off to activate signaling
pathways.
hormone
NH2
hormone
NH2
g
b
a
COOH
GDP
g
b
COOH
GTP
a
GTP
signal pathways
How G Proteins Work (cont.)
• After activating the signal pathway, the GTP is
hydrolyzed into GDP, and the alpha subunit returns to
the beta and gamma subunits at the membrane.
NH2
g
COOH
a
a
b
GDP
GTP
P
G Protein Stimulation of Cyclic AMP
• Binding of many hormones to their receptors results
in the stimulation of the second messenger, cyclic
AMP.
• G Protein involved: Gs
G Protein Stimulation of Cyclic AMP
Gs
AC
g
b
LH
a
a
b
b
LHR
a
GTP
GDP
GTP
a
b
ATP
cAMP
GTP
GTPase
GDP
Receptor-G protein Interactions
How are receptor-G protein interactions measured?
• Ligand-binding assays:
Low- affinity
High-affinity
R + G(GTP-δ-S)
RG(GDP)
GDP
GTPγS
Without GTP, both high- and low-affinity states are measured.
With GTP and Mg2+, only low-affinity state is measured, because
Agonist binding rapidly induces change from high- to low-affinity.
How Else Is Cyclic AMP Regulated?
• In addition to regulating the production of cyclic AMP,
there is also regulation of its degradation.
• Degradation of cyclic AMP is by phosphodiesterases,
which break down cyclic AMP into 5’-AMP
• Inhibitors of phosphodiesterases prolong activation of
the cyclic AMP system.
Regulation of cAMP Levels by
Phosphodiesterases
a
b
ATP
cAMP
X
PDEs
5’-AMP
GTP
(-)
PDE Inhibitor
Protein Kinase A Pathway
• Increased cyclic AMP activates protein kinase A.
• Protein kinase A (PKA) is composed of two regulatory
subunits and two catalytic subunits.
• Binding of cyclic AMP to the regulatory subunits frees the
catalytic subunits, which have kinase activity.
• PKA uses ATP to phosphorylate specific enzymes in the
cell, influencing their activity.
regulatory
catalytic
cyclic AMP
Effects of Cyclic AMP-dependent PKA on
Gene Transcription
• Cyclic AMP regulates the transcription of many genes by
increasing PKA and causing the phosphorylation of the
Cyclic AMP Response Element Binding Protein (CREB).
• CREB is a transcription factor which binds to a consensus
cyclic AMP-response element (CRE) on the 5’-flanking
region of many genes.
5’-flanking region
CAT
CRE ERE TATA
BOX
intron
exon
CREB
• The CRE has a palindromic consensus sequence:
5’-TGACGTCA-3’
3’-ACTGCAGT-5’
• Removing the CRE from cyclic AMP-responsive genes
causes a loss of regulation by cyclic AMP.
• Adding a CRE to non-cyclic AMP-responsive genes
confers responsiveness to cyclic AMP.
• CREB binds to the CRE as a dimer.
• Phosphorylation increases the dimerization of CREB,
resulting in increased transcriptional activity.
• Cells deficient in PKA cannot transcribe genes via the
CRE (phosphorylation of CREB is required)
Terminology: CRE(cyclic AMP response element);
CREB: CRE binding protein; CBP: CREB binding protein
CREM
• More recently, Cyclic AMP-Response Element
Modulators (CREMs) has been identified.
• Structurally related to CREB.
• Four isoforms exist, all the product of a single gene.
• Three isoforms block cyclic AMP-dependent gene
transcription.
• One isoform is an agonist for the CRE.
• Relative expression of isoforms is regulated in the
testis:
- immature sperm cells express antagonist form
- maturing sperm cells express agonist form
Summary of Cyclic-AMP Signaling
hormone binds receptor
Gsa activates adenylyl cyclase
adenylyl cyclase produces cyclic AMP
cyclic AMP activates PKA
cAMP-GEFs
PKA phosphorylates CREB
Ras/Rap
CREB initiates transcription
PKB/SgK
Why Make it So Complicated?
• Many steps = many places where regulation can take
place
• In addition, at several steps the signal is AMPLIFIED.
For example, activation of adenylyl cyclase produces
several cyclic AMP molecules. Each activated PKA can
phosphorylate many CREB molecules.
Influence of G Proteins on Phospholipase C
• Receptors coupled to Go activate phospholipase C,
which hydrolyzes an inositol phospholipid into
inositol triphosphate (IP3) and diacylglycerol
(DAG).
• IP3 and DAG each activate separate signaling
pathways:
IP3 activates a Ca2+ pathway.
DAG activates the protein kinase C pathway.
Actions of IP3 and Ca2+
• IP3 causes release of intracellular Ca2+ stores (ER) and
allows extracellular Ca2+ to enter the cell.
• Result: increased free cytoplasmic Ca2+.
• Ca2+ can then bind to calmodulin, activating it.
• Calmodulin influences the activity of other enzymes,
including kinases.
Go
IP3
Ca2+
calmodulin
Gq signaling pathways, Ca2+, IP3, PKC
Actions of DAG and PKC
• DAG activates calcium-dependent Protein Kinase C
(PKC), by increasing PKC’s affinity for calcium.
• PKC phosphorylates a number of enzymes at
serine/threonine residues, influencing their activity.
• PKC activates the transcription factor AP-1:
- jun/fos heterodimer
- binds to AP-1 sites on the 5’-flanking region of
genes (similar to CRE, but different!)
- binding influences gene transcription
Summary of Go Signaling
Goa
IP3
calcium
calmodulin
DAG
enzyme activity
PKC
enzyme activity
AP-1
gene transcription
Next Lecture…..
Intracellular Hormone Receptors