Hormone Signaling Pathways Lecture (Dr. Zaidi) Flashcards
Hormones
- MESSENGER Molecules
- SYNTHESIZED and SECRETED by specialized Cells called “ENDOCRINE CELLS”
- RELEASED into Extracellular Fluid or BLOOD STREAM
- Exert their influence by BINDING TO SPECIFIC RECEPTORS on the Cells of remote Target tissues
- Result in ACTIVATION OF SIGNAL TRANSDUCTION mechanisms that ultimately lead to CELL TYPE SPECIFIC RESPONSE
- Small amount of Hormone required to Alter cell Metabolism
- Effect greatly magnified via AMPLIFICATION of Signal
Hormone Signaling: A Multi Step Process
- Biosynthesis
- Storage
- Secretions
- Transport to Target Tissue/ Cells
- Recognition and Binding to Receptors
- Activation of Signal Transduction Pathway
- Relay and Amplification of Signal
- Cellular Response
- Degradation
Steps in Cell Signaling
- In RESPONSE to a STIMULUS, a Signaling cells SYNTHESIZES and SECRETES A SIGNALING MOLECULE (Lipid Soluble or Water Soluble)
- The Signaling Molecule TRANSPORTED to Target Cell, where it BINDS to a Specific RECEPTOR PROTEIN
- The Signaling Molecule- Receptor Complex ACTIVATES OR INHIBITS CELLULAR PATHWAYS that elicit a Particular Cellular Response (Metabolism, Gene Expression)
- Signal TERMINATED by REMOVAL of the Signaling Molecule and/or Receptor or INACTIVATION of the Signaling events triggered by the Signaling Molecule- Receptor Complex
Types of Signaling
1) ENDOCRINE SIGNALING:
- Signaling Molecule released by a Cell DISTANT from the Target Cell. Ex: EPINEPHRINE
2) PARACRINE SIGNALING:
- A Signaling Molecule released by One Cell Type and Diffuses to a Neighboring Target Cell of a Different Cell Type. Ex: TESTOSTERONE
3) AUTOCRINE SIGNALING:
- Singlaing Molecule acts on the Same Cell Type as the Secreting Cells themselves. Ex: INTERLEUKIN - 1
4) JUXTACRINE SIGNALING:
- Signaling Molecule stays attached to the Secreting Cell and Binds to a Receptor on an Adjacent Target Cell. Ex: HEPARIN-BIDNING EPIDERMAL GROWTH FACTOR
Hydrophilic Hormone Signaling
- Hydrophilic Hormones CANNOT PENETRATE the PLASMA MEMBRANE
- Interact with Specific RECEPTORS at the CELL SURFACE
- Ex: Epinephrine, Insulin, Glucagon, etc
- Signaling Molecule-Receptor Complex initiates production of SECOND MESSENGER Molecules Inside Cell
- This triggers the CELLULAR RESPONSE
Receptors involved in Hydrophilic Hormone Signaling:
1) G PROTEIN COUPLED RECEPTORS (GPCRs)
2) RECEPTOR TYROSINE KINASES (RTKs)
Lipophilic Hormone Signaling
- LIPOPHILIC HORMONE PASSES through PLASMA MEMBRANE of TARGET CELL
- Ex: Steroid Hormones, Thyroid Hormones and Retinoids
- Hormone (Ligand) binds to Specific RECEPTOR PROTEINS inside the Cell.
- Signaling MOLECULE- RECEPTOR COMPLEX acts as a TRANSCRIPTION FACTOR
Family of DNA Binding Transcription Factors:
1) CYTOPLASMIC RECEPTORS:
- Exist in an Inactive Complex with HSP 90. Upon Bidning to Signal HSP DISSOCIATES. The Hormone- Receptor Complex Translocates to Nucleus where it binds to a Specific DNA Sequence called the HORMONE RESPONSE ELEMENT (HRE) in the Promoter Region of Specific Genes.
2) NUCLEAR RESPONSE:
- Already present in Nucleus BOUND TO DNA. The Hormone Signaling Activated the Complex and allows for INTERACTIONS with Additional Proteins
BOTH REGULATE THE TRANSCRIPTION OF SPECIFIC GENES**
Lipophilic (“Lipid Loving”)
SIGNALING MOLECULES:
- Steroid Hormones: Progesterone, Estradiol, Testosterone, Cortisol, Aldosterone, Vitamin D
- Thyroid Hormone: Thyroxine
- Retinoids: Retinol, Retinoid Acid
RECEPTOR LOCATION AND TYPE:
- Found in the CYTOPLASM and NUCLEUS
- Family of DNA-BINDING TRANSCRIPTION FACTORS
Hydrophilic (“Water Loving”)
SIGNALING MOLECULES:
- Amino Acid Derived: Histamine, Serotonin, Melatonin, Dopamine, Norepinephrine, Epinephrine
- From Lipid Metabolism: Acetylcholine
- Polypeptides: Insulin, Glucagon, Cytokines, Thyroid Stimulating Hormone
RECEPTOR LOCATION and TYPE:
- Found on the Surface of Plasma Membranes
- Includes Transmembrane proteins such as G Protein- Coupled Receptor and Receptor Tyrosine Kinases
Lipophilic and Hydrophilic Medications
1) HYDROPHILIC MEDICATIONS:
- Have a SHORT HALF LIFe (Seconds to Minutes)
- For Ex: EPINEPHRINE contained in Autoinjectors used to Treat Severe ACUTE ALLERGIC Reaction that may lead to ANAPHYLACTIC SHOCK. Given at TIME OF NEED!!!
2) LIPOPHILIC MEDICATIONS:
- Have LONG HALF LIFE (Hours to Days)
- For Ex: ORAL CONTRACEPTIVES contain Ethinyl Estradiol, a derivative of Estradiol. NEED TO TAKE DIALY!
GPCR Signaling
- TRIMERIC G PROTEINS contain THREE SUBUNITS (Alpha, Beta, and Gamma)
- An INACTIVE G PROTEIN has Guanosine Diphosphate (GDP) bound to its Alpha Subunit, which is attached to the Beta and Gamma Subunits
- To become ACTIVE, the G Protein must Exchange its GDP FOR GUANOSINE TRIPHOSPHATE (GTP)
- This occurs via the action of a GUANINE NUCLEOTIDE EXCHANGE FACTOR (GEF)
- The active, GTP Bound Alpha Subunit separates from Beta and Gamma Subunits
- To return to its INACTIVE STATE, the INTRINSIC GTPASE ACTIVITY of the G Protein Hydrolyzes its bound GTP into GDP and Phosphate (Pi)
- This action is Accelerated by a GTPASE- ACTIVATING PROTEIN (GAP)
GPCR Signaling Variations
1) Gs: Stimulates ADENYLATE CYCLASE
2) Gi: INHIBITS ADENYLATE CYCLASE
3) Gt: STIMULATES HYDROLYSIS of cGMP!!!!
G Protein Coupled Receptor (GPCR) Signaling
- Binding of a Singlaing Molecule to a GPCR causes Activation of Gs or Gi, which in turn STIMULATES or INHIBITS ADENYLATE CYCLASE
- Activated AC Converts ATP to cAMP, a SEOND MESSENGER that regulates the activity if Protein Kinase A (PKA). Enzymatically INACTIVE PKA exists as a TETRAMERIC COMPLEX Containing Two Regulatory Subunits (R) and Two Catalytic Subunits. Binding of cAMP to the Regulatory Subunits causes the Complex to DISSOCIATE.
- The FREE, ACTIVE Catalytic Subunits Phosphorylate Target Proteins. cAMP is Hydrolyzed into AMP by the Action of PHOSPHODIESTERASE (PDE), an Enzyme that is INHIBITED by CAFFEINE
- Light triggers the GPCR- mediated activation of Gt, which, in turn, stimulates the Hydrolysis of CYCLIC GUANOSINE MONOPHOSPHATE (cGMP) by cGMP PDE
G Protein- Coupled Receptor (GPCR) Signaling via Gq, Phospholipase C (PLC) and Protein Kinase C (PKC)
- Binding of a Singlaing Molecule to a GPCR Triggers the ACTIVATION OF Gq, which stimulates the CLEAVAGE OF PHOSPHATIDYL INOSITOL 4,5 BISPHISPHATE (PIP2) by PHOSPHOLIPASE C (PLC) to yield INOSITOL 1,4,5- TRIPHOSPHATE (IP3) and DIACYLGLYCEROL (DAG)
- IP3 causes the RELEASE OF CA2+ from the Endopalsmic/ Sarcoplasmic Reticulum (ER/ SR) into the Cytosol
- An Increase in Cytoplasmic CA2+ causes the Cytosolic Enzyme PROTEIN KINASE C (PKC) TO TRANSLOCATE TO THE PLASMA MEMBRANE, where it is activated by DAG
- Ca2+ also binds to the Cytosolic Protein CALMODULIN, forming a Complex that activates Ca2+-Calmodulin-Dependent Proteins, which include CA2+-CALMODULIN-DEPENDENT PROTEIN KINASE (CaM Kinase) and MYOSIN LIGHT CHAIN (MLC) KINASE
- Activated CaM Kinase Phosphorylates Target Proteins to alter their activities, whereas activated MLC Kinase Phosphorylates MYOSIN LIGHT CHAINS, causing SMOOTH MUSCLES TO CONTRACT
Gs GPCR with EPINEPHRINE
Physiological Response:
- RELAXATION of BRONCHIAL and INTESTINAL Smooth muscle
- CONTRACTION of Heart Muscle
- INCR Breakdown of Triacylglycerides in Adipose Tissue
- INCR Breakdown of Glycogen in Liver and Muscle
- INCR Glycolysis in Muscle
***EPINEPHIRNE NON-SELECTIVE AGONIST OF ALL ADRENERGIC RECEPTORS (MAJOR SUBTYPES Alpha1, Alpha2, Beta1, Beta2, Beta3) UNDERGO MULTIPLE GPCR SIGNALING PATHWAYS!
Gs GPCR with HISTAMINE
Physiological Response:
- BRONCHOCONSTRICTION and SYMPTOMS of ALLERGIC REACTIONS (Itchy, Watery Eyes)
Gi GPCR with Epinephrine/ Norepinephrine
- CONSTRICTION of Smooth Muscle
Receptor Tyrosine Kinase
- ISNULIN Hormoen (Ligant) bidns to its Receptor (RTK) to regulate GLUCOSE METABOLSIM
1) RAS- DEPENDENT Signaling
2) RAS- INDEPENDENT Signaling
Primary Structure of Insulin
- Insulin composed of Two Peptide Chains referred to as the A Chain and B CHAIN
- They are linked together by TWO DISULFIDE BRIDGES, and an Additional DISULFIDE is formed WITHIN the A Chain
- In most Species, the A Chain consists of 21 Amino Acids and the B CHAIN of 30 Amino Acids
Secondary and Tertiary Structure of Insulin
- High Resolution Model of 6 INSULIN Molecules assembled in a HEXAMER
- Has a 3 FOLD Symmetry with ZINC in the Center connected to the Polypeptides via HISTIDINES
*****INACTIVE INSULIN is stored in the BODY as a HEXAMER, while the ACTIVE FORM is the MONOMER
Insulin Synthesis and Secretions
- PROPROINSULIN mRNA Transcription and Translation (Protein Synthesis)
- PROPROINSULIN (110 Amino Acids) has a N Terminal Hydrophobic Signal Peptide which interacts with the Signal Particle that directs it to ER
- Translocated into Lumen of ER
- Cleaved by a Protease to for PROINSULIN
- Proinsulin folds into Correct Conformation and 3 Disulfide Added. ER Chaperone Protein help in this
- Transported into GOLGI Apparatus
- Packaged into Immature CLATHRIN-COATED Granules
- Cleaved by PROTEASES to form INSULIN and C PEPTIDE
(ENDOPEPTIDASES aka Prohormone Converts (PC1 and 1) and a Carboxypeptidase) - The Immature Granules then become MATURE Granules containing HEXAMETRIC Crystaliized Insulin (Three Dimers). Also contain Amylin and other B Cell Secretory Products
- INSULIN + C PEPTIDE released TOGETHER
Regulation of Insulin Synthesis and Secretion
- After GLUCOSE Stimulation INSULIN Granules exhibit 2 Characteristics:
1) Rapidly INITATED but TRANSIENT
2) SUSTAINED
- The Granules are divided into 2 Different Pools
- (1) A LIMITED POOL of Granules (5%) ready for IMMEDIATE Release and is referred to as the “READILY RELEASABLE POOL” (RRP), which account for the FIRST Release Phase
- (2)Most of the Granules (95%) belong to a RESERVE POOL responsible for the SECOND-PHASE of Insulin Secretion. Granules in this pool must undergo MOBILIZATION before they can GAIN Release Competence
RAS Dependent Insulin Signaling
- Insulin binds to RTK which exists as a Performed Dimer
- Causes Autophosphorylation of the Receptors TYROSINE Residues
- Phosphotyrosine residues recognized and bound by Protien called INSULIN RECEPTOR SUBSTRATE 1 (IRS-1)
- IRS-1 Phosphorylated on its TYROSINE by INSULIN Receptor
- Phsophrylated IRS-1 recognized and bound by Adaptor Protein GRB-2 initiating the Transcription of RAS and the MAP Kinase Pathway
- Results in Phosphorylation of Nuclear Proteins that INCREASE Transcription of Glucokinase
- GLUCOKINASE phosphorylated Glucose in the First Step of Glycolysis and Glycogen Synthesis
- IRS-2 similar to IRS-1 but has different Specificity for Adaptor Proteins
RAS- Independent Insulin Signaling
- INSULIN binds to RTK which exists as a PERFORMED DIMER
- Causes AUTOPHOSPHORYLATION of the Receptor’s Tyrosine Residues
- PHOSPHOTYROSINE residues recognized and bound by IRS-1
- IRS-1 Phosphorylated on its Tyrosine by Insulin Receptor
- Phosphorylated IRS-1 recruits PHOSPHOIONOSITIDE 3 KINASE (PI3Kinase)
- PI3 KINASE Phosphorylates Phosphoinositides to form Phosphatidyl Inositol 3,4- Bisphosphate and Phosphoinositol 3, 4, 5 Triphsopahte (PIP3)
- These Membrane bound Phosphoinositides act as Second Messengers. Stimulate the Recruitment of PROTEIN KINASE B (PKB) to the membrane and its Activation via PHOSPHORYLATION
- Active PKB (serine Threonine Kinase) also known as AKT PHOSPHORYLATES and alters the activity of several Intracellular Proteins
- Has stimulatory effects on Glucose uptake and Storage
- For example, PKB plays a role in the Insulin- Induced movement of GLUCOSE TRANSPORTER (GLUT 4) form Cytoplasm to Plasma Membrane of Muscle and Adipose Cells
- PKB also promoted Glycogen Synthesis by PHOSPHORYLATING and INHIBITING GLYCOGEN SYNTHASE KINASE 3 (GSK-3)
Insulin Resistance
- Quantifiable Parameter: Measured as the AMOUNT OF GLUCOSE CLEARED FROM THE BLOOD IN RESPONSE TO A DOSE OF INSULIN
- Failure of Normal Amounts of Insulin to ELICIT the expected response is referred to as INSULIN RESISTANCE
- Mechanism not fully understood
- DEFECTS IN INSULIN SINGLAING (Receptor Activation to Translocation of GLUT4 transporter in ADIPOSE and SKELETAL MSUCLE TISSUE)
- > 75 different Mutations in INSULIN RECEPTOR Identified. Impair Action of the Receptor
- Defects in Insulin binding Domain on the Extracellular Side- SEVER RESISTANCE
- INCREASED PHOSPHORYLATION OF SERINE (Via Ser/ The Kinase) INSTEAD OF TYROSINE in the IR and the IRS- Inhibits ACTIVATION AND SIGNALING
- PHOSPHORYLATION of IRS NEEDED For RECRUITMENT of PI3 KINASE but Ser/ The PHOSPHORYLATION APPEARS to INACTIVATE the IRS 1 and 2, leading to DEGRADATION!!!!!
- Ser/ Thr KINASE ACTIVATED by CYTOKINES, FREE FATTY ACIDS, DAG, CERAMIDE, INFLAMMATORY MOLECULES!!!
Effects of Insulin, Glucagon, Epinephrine, and Cortisol on Glucose Metabolism
- Under Fed conditions, Insulin LOWER BLOOD GLUCOSE by promoting Glycogen Synthesis, stimulation GLYCOLYSIS, and INHIBITING the activity and Synthesis of Enzymes for GLUCONEOGENESIS
- A DEFICIENCY in INSULIN PRODUCTION (Type I Diabetes) or INSENSITIVITY to INSULIN (Type 2 Diabetes) results in ELEVATED BLOOD GLUCOSE
- Under conditions of Glucose Deficiency, PANCREAS release Glucagon
- Glucagon INCREASES Blood Sugar by promoting breakdown of Glycogen in Liver and Inhibiting Breakdown of Glycogen by promoting Glucagon Secretion
- When the GLYCOGEN stores depleted, then Steroid Hormone Cortisol stimulates GLUCONEOGENSIS by Inducing Transcription of Enzymes involved in this Pathway
Lipophilic Hormone Signaling Pathways
NUCLEAR RECEPTORS (NR)
- Large Superfamily of Receptors- Include (a) Nuclear Hormone Receptors and (b) Orphan Receptors
- Classification based on Liands they bind
1) CLASSIC STEROID NUCLEAR RECEPTORS: Ligands are Lipophilic Hormones. Ex: Glucocorticoids GR), Mineralocorticoids (MR), Estrogen (ER), Progesterone (PR) and Androgens (AR)
2) ORPHAN RECEPTORS: Other group of Receptors discovered by DNA Sequencing (Ligands Unknown) - Ligands for Several Have been discovered since are “ADOPTED” Orphan Receptors Now. Ex: Retinoids, Thyroid Hormones, Vitamin D, Xenobiotics, and Androstane
Nuclear Receptors Cont
- NRs may be localized in NUCLEUS or in CYTOSOL
- After Ligand Binding they TRANSLOCATE TO NUCLEUS and Influence GENE EXPRESSION
- Serve as Receptors and Effectors for the Signal
- Important Drug Targets
- Diseases involved aberration in NR Signaling: Reproductive Disorders, Cancer, Diabetes, Inflammation, Cardiovascular Disorders, Obesity etc
Molecule Structure of NRs
Same Architecture (3 Major Domains) 1) ACTIVATION FUNCTION 1 DOMAIN (AF1)
2) DNA BINDING DOMAIN (DBD)
3) LIGAND BINDING DOMAIN (LBD)
- AF1 is INDEPENDENT of Ligand Binding, can Modify the Conformation of the Entire Receptor
- DBD Highly conversed, binds to Regulatory sequences of DNA called HORMONE RESPONSE ELEMENT (HRE), Upstream of Target Gene
- LBD binds to various Molecules (AGONIST or ANTAGONIST) which regulates Ligand-Dependent Activation of Receptor. Upon Ligand Binding a region within LBD (AF2) undergoes CONFORMATIONAL CHANGES allowing Recruitment and Biding of COACTIVATORS or COREPRESSORS that Regulate Transcription
- NRs exist as HOMO or HETERODIMERS
Estrogen Receptor (ER)
- Two MAIN TYPES (ER Alpha and ER Beta)
- Products of 2 Separate Genes located on DIFFERENT CHROMOSOMES
- Structure of the 2 Similar with some differences
- Both are ESTROGEN-DEPENDENT TRANSCRIPTION FACTORS
- Differential Tissue Expression, Influence lots of Target Genes
- Mediate a Variety of Differential Biological Effects
- Knock out mice of the 3 forms have Different Phenotypes
- ER Beta can SUBSTITUTE for ER Alpha ins poem Biological Pathways
- Selective Ligands have been developed but not in Clinical Use yet
ERs
- ER ALPHA, the first discovered, is EXPRESSED MOST ABUNDANTLY in the Female Reproductive Tract (especially in the UTERUS, VAGINA, and OVARIES). Also in the MAMMARY GLAND, HYPOTHALAMUS, ENDOTHELIAL CELLS, and VASCULAR SMOOTH MUSCLE
- ER BETA is expressed most Abundantly in the PROSTATE and OVARIES, with LOWER EXPRESSION IN LUNG, BRAIN, BONE, and VASCULATURE
- Many Cells express BOTH ER ALPHA and ER BETA
- Dimerize to forma either Homodimers or Heterodimers
- FINAL BIOLOGICAL EFFECT MEDIATED BY THE RAITIO OF THE 2 FORMS!!!!!!!
- Disruption in the Ratio leads to Disease
- Most Ligands have Differential Affinity for the 2 forms
- Both ER Alpha and ER Beta are expressed in BREAT CANCERS, although ER Alpha is believed to be the PREDOMINANT form RESPONSIBLE for GRWOTH REGULATION
Molecular Mechanism of Activation of Nuclear Estrogen Receptor
GENOMIC EFFECTS
- ER exist as a MONOMER within the NUCLEUS
- The Ligand (Estrogen, Agonist such as 17 Beta- Estradiol) Binds to the Receptor.
- Causes a LIGANG-DIRECTED Conformational Change which Facilitates DIMERIZATION and INTERACTION with Specific Estrogen Element (ESE) sequences in DNA
- The ER-DNA Complex recruits CO-ACTIVATORS such as SWI/ SNF that Modify CHROMATIN STRUCTURE, and Co-Activators such as Steroid- Receptor Co-Activator- 1 (SRC-1)
- SRC-1 has HISTONE ACETYLTRANSFERASE (HAT) ACTIVITY THAT FURTHER ALTERS CHROMATIN STRUCTURE
- Chromatin remodeling Facilitates Binding of other Co-Activators (Ex: p300 and the TRAP Complex) to Promoter of Target Genes
- Additionally, it RECRUITS proteins that Comprise the GENERAL TRANSCRIPTION APPARATUS (GTA), leading to mRNA Synthesis
Some evidence that ER LOCATED in CYTOSOL. ESTROGEN BINDING CAUSES DIMERIZATION AND TRANSLOCATION TO THE NUCLEUS!!!!!!!!**
Action of Tamoxifen
- ANTAGONISTS such as Tamoxifen (T) also to ER but produce a Different Receptor Conformation
- This conformation also Facilitates Dimerization and Interaction with DNA
- A Different set of Proteins called Co-Repressors, such as Nuclear-Hormone Receptors Co-Repressor (NcoR), are recruited to the Complex
- NcoR further recruits proteins such as HISTONE DEACETYLASE I (HDAC1) that act on Histone Proteins to Stabilize Nucleosome Structure and Prevent Interaction with the GTA
ER Signaling Pathways
NON-GENOMIC
- Some ERs located in the Plasma Membrane
- Some present in CAVEOLAE (Cholesterol-enriched Domains in the Plasma Membrane)
- GPCR and RTK Type
- Effects Mediated through Metabolic Changes as well as changes in Gene Expression
