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 ReceptorReceptor
ExtracellularExtracellular IntracellularIntracellular
Which hormones Which hormones should act through should act through extracellular receptor intracellular receptor and why? 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 ReceptorReceptor
ExtraExtracceelllularlular IntracellularIntracellular
SecondSecond messengermessenger systemsystem 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 C- protein- kinase C pathway ReceptorReceptor
ExtracellularExtracellular IntracellularIntracellular
SecondSecond messengermessenger sysystemstem
AdedylAdedyl cyclasecyclase ccAMPAMP
Calcium-dependentCalcium-dependent phphospholospholiippaseaseCC 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 Guanyl cyclase cyclase synthesize
Second cAMP cGMP messengers
activate Protein activate kinase A Hormone action via H extracellular receptor R GTP Adenyl cyclase ATP G-Protein C-AMP Protein kinase A Phosphoprotein phosphatase protein
Phosphorylated Cellular protein metabolism Hormones act via cAMP second messenger system
Glucagon Secretin Calcitonin Parathyroid hormone Thyrotropin (TSH) ACTH LHRH TRH LH FSH Vasopressin 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 zPyruvate kinase : glycolysis and gluconeogenesis zGlycogen synthase : glycogen synthesis z3-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-responsive- element 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 C- protein- kinase C pathway ReceptorReceptor
ExtraExtracceelllularlular IntracellularIntracellular
SecondSecond messengermessenger sysystemstem
AdedylAdedyl cyclasecyclase ccAMPAMP
CalcCalcium-dependeium-dependenntt phospholipasephospholipase CC The calcium-dependent phospholipase C-protein kinase C pathway 2. The calcium-dependent phospholipase C- protein kinase C pathway
Hormone +receptor activates phospholipase C to
split phosphatidylinositol in the cell membrane to
inositol phosphate (IP3) and diacylglycerol (DAG)
2+ IP3 increases intracellular Ca
2+ Ca and DAG activate Protein kinase C Calcium-dependent phospholipase C- protein kinase C pathway
2+ Ca 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 diacylglycerol (DAG) (IP3)
increase activate intracellular Ca2+ Protein kinase C Calcium-dependent phospholipase C- protein kinase C pathway (cont.)
2+ Ca 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 C- protein kinase C pathway (cont.)
2+ IP3 increases intracellular Ca , 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
Phosphatidyl R phosphatidylinositol GTP -4,5-biphosphate
G-Protein IP3 IP4
Phospholipase C DAG Ç Ca2+
Protein kinase A Phosphoprotein phophatase protein Cellular Phosphorylated metabolism protein GnRH : Example of hormone action via calcium- dependent phospholipase C-protein kinase C pathway
GnRH produced by hypothalamus and caused the release of LH & FSH from pituitary gland
2+ GnRH increases intracellular Ca and affects inositol metabolism
2+ Increase intracellular Ca 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 ReceptorReceptor
ExtraExtracceelllularlular IntracellularIntracellular
SecondSecond messengermessenger sysystemstem
AdedyAdedyll cycyclaseclase ccAMAMPP
CalcCalcium-dependeium-dependenntt phospholipasephospholipase CC Interaction of cAMP pathway and Ca2+ pathway Interaction of cAMP & Ca2+ 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 ReceptorReceptor
ExtracellularExtracellular IntracellularIntracellular
SecondSecond messengermessenger sysystemstem
AdedylAdedylcyclasecyclaseccAMPAMP
Calcium-Calcium-dependependdentent phospholipasephospholipaseCC 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, 1,25-
hydroxy 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 Binding to receptor (H-R), release off HSP90 z H-R translocates to nucleus interact with hormone-responsive elements on specific genes to affect DNA transcription z Expose template sites on DNA, either directly or by influencing pre-existing repressor molecules, to increase initiation sites for RNA polymerase and increase transcription z Take longer time (hours) than peptide hormones Mechanism of action of steroid hormones
www.sutree.com/how-to/737/Biology,-7th-Editio...
http://highered.mcgraw-hill.com/olc/dl/120109/bio46.swf
http://highered.mcgraw-hill.com/olc/dl/120109/bio46.swf Structural and functional domains of nuclear receptors z Ligand-binding domain : z where hormone binds to receptor z Sequence diversity gives specificity of the receptor to hormone z DNA-binding domain : z where H-R binds to HREs to stimulate transcription z 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 cAMP- responsive 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.mcgraw- hill.com/sites/0072943696/student_view0/chapter10/animation_ _positive_and_negative_feedback__quiz_1_.html Membrane bound receptors http://highered.mcgraw- hill.com/sites/0072943696/student_view0/chapter10/animation__membrane- bound_receptors__g_proteins__and_ca2__channels.html