Download Receptors and Hormone Action

Receptors and
Hormone Action
Suporn Katawatin
Department of Animal Science
Faculty of Agriculture
Khon Kaen University
„
Hormones must binding to receptors at target cells
to do their works
• Receptors
Extracellular receptors : cell membrane
Intracellular receptors : inside the cell
Receptor
Receptor
Extracellular
Extracellular
Which hormones
should act through
extracellular receptor
and why?
Intracellular
Intracellular
Which hormones
should act through
intracellular receptor
and why?
Extracellular receptors
Extracellular receptors
„
Large molecules located on the outer surface of
plasma membrane in target tissues
„
e.g. Insulin receptor
…
…
MW 200-400 kDa
two α subunits of 130 kDa and two β-subunits of 90 kDa
Insulin Pathway
Evidences that there are extracellular receptors
„
Antibodies against receptor can block hormone action
„
Limited proteolysis of intact cells, expected to destroy receptor,
remove hormone response
„
Coupling hormone to large molecule that cannot enter cell, the
effect of hormone is still present
„
Subcellular fractionation demonstrate presence of receptor in
plasma membrane
Hormone + extracellular receptor
„
activate intracellular enzyme systems via
synthesis of intracellular
second messengers
to alter cell function
Receptor
Receptor
Extracellular
Extracellular
Second
Second messenger
messenger
system
system
Intracellular
Intracellular
Second-messenger System
Second-messenger System
There are two pathways
1.
Adenyl cyclase-cAMP-protein-kinase pathway or guanyl
cyclase-cGMP-protein-kinase pathway
2. Calcium-dependent phospholipase Cprotein- kinase C pathway
Receptor
Receptor
Extracellular
Extracellular
Intracellular
Intracellular
Second
Second messenger
messenger system
system
Adedyl
Adedyl cyclase
cyclase cAMP
cAMP
Calcium-dependent
Calcium-dependent
phospholipase
phospholipase CC
1. Adenyl cyclase-cAMP-protein-kinase pathway or
guanyl cyclase-cGMP-protein-kinase pathway
z Hormone + receptor activates enzyme adenylate cyclase or guanalate
cyclase, which synthesize second messenger : either cAMP or cGMP
z Second messenger then activates
Protein kinase A
z Protein kinase A, then, phosphorylated protein and alter cellular response
Secondary messenger pathway
Hormone + Receptor
activate
Adenyl
cyclase
synthesize
cAMP
Guanyl
cyclase
Second
messengers
cGMP
activate
activate
Protein
kinase A
Hormone action via
extracellular receptor
H
R
GTP
ATP
G-Protein
Phosphoprotein
phosphatase
Adenyl cyclase
C-AMP
Protein kinase A
protein
Phosphorylated
protein
Cellular
metabolism
Hormones act via cAMP
second messenger system
Glucagon
Secretin
Calcitonin
Thyrotropin (TSH)
LHRH
LH
Vasopressin
Parathyroid hormone
ACTH
TRH
FSH
Chorionic gonadotropin
Substrates for cAMP-dependent
protein kinase
z Triglyceride lipase : lipolysis
z Phosphorylase β kinase : glucogenolysis
z Cholesterol ester hydrolase : steroidogenesis
z Fructose1, 6-diphosphatase : gluconeogenesis
z These Substrates (enzymes) are
Activated By Phosphorylation
Substrates that are
inactivated by phosphorylation
z Pyruvate kinase : glycolysis and
gluconeogenesis
z Glycogen synthase : glycogen synthesis
z 3-hydroxy-3methylglutaryl-CoA reductase :
cholesterol biosynthesis
1.1 Guanyl cyclase-cGMP-dependent protein
kinase pathway
z Similar to cAMP system, but may act in
opposition to cAMP e.g.
{cAMP-dependent kinases results in smooth muscle
relaxation
{cGMP-dependent kinases results in smooth muscle
contraction
z Level of cGMP are normally 10-50 times
lower than cAMP
Action of cAMP
z Activating protein kinase A, then
z Phosphorylate intracellular proteins
z Cause immediate cellular response : modify
metabolic pathway, regulation of ion flows,
muscle contraction
However, cAMP can also affect
gene transcription
cAMP can affect gene transcription
z E.g. Protein kinase A activate cAMP-responsiveelement binding protein (CREB), or modify structural
proteins in chromatin
z Activated CREB binds to specific cAMP-responsive
elements in the regulatory regions of certain genes
to activate gene expression
Second-messenger System
There are two pathways
1. Adenyl cyclase-cAMP-protein-kinase pathway or
guanyl cyclase-cGMP-protein-kinase pathway
2. Calcium-dependent phospholipase Cprotein- kinase C pathway
Receptor
Receptor
Extracellular
Extracellular
Intracellular
Intracellular
Second
Second messenger
messenger system
system
Adedyl
Adedyl cyclase
cyclase cAMP
cAMP
Calcium-dependent
Calcium-dependent
phospholipase
phospholipase C
C
The calcium-dependent
phospholipase C-protein
kinase C pathway
2. The calcium-dependent phospholipase Cprotein kinase C pathway
„
Hormone +receptor activates phospholipase C to
split phosphatidylinositol in the cell membrane to
inositol phosphate (IP3) and diacylglycerol (DAG)
„
IP3 increases intracellular Ca2+
„
Ca2+and DAG activate
Protein kinase C
Calcium-dependent phospholipase Cprotein kinase C pathway
„
Ca2+is the primary intracellular
effector in this system
Calcium-dependent phospholipase C-protein kinase C pathway
activate
Hormone
phospholipase C
+
To split
Receptor
phosphatidylinositol
inositol phosphate
(IP3)
diacylglycerol
(DAG)
activate
increase
intracellular Ca2+
Protein kinase C
Calcium-dependent phospholipase Cprotein kinase C pathway (cont.)
„
Ca2+ activates calcium-dependent protein kinase C,
„
Phospholipase C catalyses the hydrolysis of
phosphatidylinositol-4,5-biphosphate to produces
inositol-1,4,5-phosphate (IP3) & Diacylglycerol
(DAG)
Calcium-dependent phospholipase Cprotein kinase C pathway (cont.)
„
IP3 increases intracellular Ca2+, by activating
Ca channels at ER and cell membrane
„
DAG activates protein kinase C, by increasing
its affinity for Ca2+
„
Protein kinase C phosphorylates cellular
proteins to regulate their activities
H
R
Phosphatidyl
phosphatidylinositol
-4,5-biphosphate
GTP
G-Protein
IP3
Phospholipase C
DAG
IP4
Ç Ca2+
Protein kinase A
Phosphoprotein
phophatase
protein
Phosphorylated
protein
Cellular
metabolism
GnRH : Example of hormone action via calciumdependent phospholipase C-protein kinase C
pathway
„
GnRH produced by hypothalamus and caused
the release of LH & FSH from pituitary gland
„
GnRH increases intracellular Ca2+ and affects
inositol metabolism
„
Increase intracellular Ca2+ causes a release of
LH
G-Protein
„
Receptors interact with adenyl cyclase or phospholipase C
via G-protein
„
G-protein is activated by binding GTP and inactivated when
GTP converted to GDP by GTP-ase
„
G-protein act to couple extracellular receptors for hormones,
neurotransmitters, odorants and photons of light to effector
molecules, ie. ion channels or enzymes that generate second
messenger molecules
Calmodulin
„
Heat-stable globular protein, 16 kDa
„
Calcium-dependent regulatory protein
„
Controlls intracelllar Ca2+ and binds 4 Ca2+ to form active
complex
„
The complex acts as an allosteric regulator of protein kinase C
and other enzymes
„
Also controls activity of cellular filamentous organells, via actin
& myosin, responsible for cell motility, exoplasmic secretion &
chromosome movement
Receptor
Receptor
Extracellular
Extracellular
Intracellular
Intracellular
Second
Second messenger
messenger system
system
Adedyl
Adedyl cyclase
cyclase cAMP
cAMP
Calcium-dependent
Calcium-dependent
phospholipase
phospholipase C
C
Interaction of cAMP pathway
and Ca2+ pathway
Interaction of cAMP &
„
2+
Ca
pathways
Ca2+calmodulin complex bind and activate phosphodiesterase to
decrease cAMP
„
Protein kinase A , which is activated by cAMP, can phosphorylate
Ca2+ channels & pumps to affect intracellular Ca2+ level
„
Protein kinase A can change protein kinase C activity by
phosphorylation
„
Protein kinase A & protein kinase C can phosphorylate different
sites on the same protein, so that its activity is regulated by both
cAMP and Ca2+
Let’s take a break
Tyrosine kinase receptors
• Special receptor as having a kinase
domain as part of the receptor
• not use second messenger system
to activate protein kinase
•E.g. EGF, IGF-1, PDGF, NGF
Tyrosine kinase receptors
Receptor
Receptor
Extracellular
Extracellular
Intracellular
Intracellular
Second
Second messenger
messenger system
system
Adedyl
Adedyl cyclase
cyclase cAMP
cAMP
Calcium-dependent
Calcium-dependent
phospholipase
phospholipase CC
Intracellular receptor
Suporn Katawatin
Department of Animal Science
Khon Kaen University
Intracellular receptor
z
Steroid and thyroid hormones
z
Cytoplasmic receptor: glucocorticoids,
mineralocorticoids, androgens
z
Nucleus receptors : thyroid hormones,
estrogen, progesterone, retinoic acid, 1,25hydroxy vitamin D3
Steroid hormone receptors
z
Act as transcription factors to regulate
transcription of the target genes
z
Steroid hormone receptors move between
nucleus and cytoplasm
z
In the absent of hormone, steroid receptors
bound to HSP90 (except thyroid hormone,
retinoic acid, Vit. D)
Steroid hormone can affect the
response to other hormone, through
synthesis of receptors or protein kinases
to increase hormone response
or phosphoprotein phosphatases,
which are antagonistic to cyclic nucleotide
actions
Mechanism of action of steroid hormones
z
z
z
z
Binding to receptor (H-R), release off HSP90
H-R translocates to nucleus interact with
hormone-responsive elements on specific
genes to affect DNA transcription
Expose template sites on DNA, either directly
or by influencing pre-existing repressor
molecules, to increase initiation sites for RNA
polymerase and increase transcription
Take longer time (hours) than peptide
hormones
Mechanism of action of steroid hormones
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Structural and functional domains of
nuclear receptors
z
Ligand-binding domain :
z
z
z
where hormone binds to receptor
Sequence diversity gives specificity of the
receptor to hormone
DNA-binding domain :
z
z
where H-R binds to HREs to stimulate
transcription
comprised of 60-70 aa in “zinc fingers”
Structural domains of intracellular receptors
Hormone-responsive elements
z
Specific in hormone target genes
z
Identification of these sequences in a gene
suggests that hormones regulate the gene
z
Sequences usually in 5’ regions, but may
also be in introns or 3’ region
Experiment to illustrate that steroid hormone receptor
binding increases RNA polymerase initiation sites in target
genes
z
Increase in RNA polymerase initiation sites caused
by binding of steroid H-R complex to DNA can
be demonstrated using inhibitors of free RNA
polymerase, rifampicin or a-amanitin
z
(Number of copies of RNA transcribed is a
measure of number of initiation sites on
chromatin)
• RNA polymerase added
to chromatin to bind
initiation sites, then
• rifampicin added to
bind and inhibits excess
RNA polymerase
•Nucleotides added to
start transcription,
•Result : only one copy
is made as after that
RNA polymerases are
inhibited by rifampicin
Integration of peptide and
steroid hormone action
„
Steroid hormones can cause rapid effects
by acting at the cell surface and not only
longer-term effects on gene transcription
by binding to intracellular receptors
For example
„
Plasma sex hormone binding globulin (SHBG), transport androgens and
estrogens in blood, thus regulates amount of free sex hormones released to
target cells
„
SHBG binds to membrane receptor and then binds free steroids, to activate
cAMP second messenger system: Thus SHBG modulate effects of sex steroids
acting on receptors within the target cells
„
Steroids bind to SHBG but not activate second messenger system act as
antagonists
„
Mineralocorticoids, glucocorticoids, vitD3, thyroid hormones are also known to
be rapid non-genomic effects steroids (Falkenstein et al., 2000)
„
Fortunati (1999)
Steroid hormones can also affect activity
of protein hormones
„
Stimulating the synthesis of protein hormone receptors
on membrane surface
„
Affecting synthesis of hormone kinases or other
intracellular protein so that involves in the action of
peptide hormones
Effects of cAMP on gene transcription
„
Phosphorylation & activation of cAMP-responsive-element
binding protein (CREB) by protein kinase A or by
modification of the structural proteins in chromatin
„
Many hormone-responsive genes have specific cAMPresponsive elements in their regulatory regions, thus
activated CREB binds to these regions to activate gene
expression
„
Stryer (1995); Nelson and Cox (2000)
Website
„
Feedback system
http://highered.mcgrawhill.com/sites/0072943696/student_view0/chapter10/animation_
_positive_and_negative_feedback__quiz_1_.html
„
Membrane bound receptors
http://highered.mcgrawhill.com/sites/0072943696/student_view0/chapter10/animation__membranebound_receptors__g_proteins__and_ca2__channels.html