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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 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 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