Receptors and Action

Suporn Katawatin

Department of Animal Science

Faculty of Agriculture

Khon Kaen University „ must binding to receptors at target cells to do their works

• Receptors

Extracellular receptors :

Intracellular receptors : inside the cell ReceptorReceptor

ExtracellularExtracellular IntracellularIntracellular

Which hormones Which hormones should act through should act through extracellular and why? and why? Extracellular receptors Extracellular receptors

„ Large molecules located on the outer surface of plasma membrane in target tissues

„ e.g. 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 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--kinase pathway or guanyl

cyclase-cGMP-protein-kinase pathway

2. -dependent 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

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 Hormones act via cAMP second messenger system

Glucagon Secretin Thyrotropin (TSH) ACTH LHRH TRH LH 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 () are Activated By Phosphorylation Substrates that are inactivated by phosphorylation zPyruvate kinase : glycolysis and gluconeogenesis zGlycogen synthase : 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 relaxation {cGMP-dependent kinases results in smooth z Level of cGMP are normally 10-50 times lower than cAMP Action of cAMP z Activating protein kinase A, then z Phosphorylate intracellular 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 - 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 in the cell membrane to

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,

, odorants and photons of light to effector

molecules, ie. ion channels or enzymes that generate second

messenger molecules

„ 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 & 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 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 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 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 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 -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 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