Metabolic Endocrinology - Hormone Action
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How can pathways be divided (based on signal receptors)
- 2 ways:
- Pathways with cell-surface receptors
- Pathways with intracellular receptors
How do cell-surface receptors work?
- Hydrophilic signal molecule binds to cell-surface receptor, which transports it across the plasma membrane
- Soluble in blood, cannot cross lipid membrane because they are not lipid soluble
How do intracellular receptors work?
- As long as hormone is bound to carrier protein it is soluble
- Small hydrophobic signal molecule
- Enter through lipid membrane
What is the base structure of a cell surface receptor
- Ectodomain - NH2 and glycosylations
- Hydrophobic transmembrane domain - 25 AA needed to cross memebrane
- Cytoplasmic domain
Where does the signal bind on a cell-surface receptor?
- Ectodomain - NH2 end of peptide
- Rich in cystei residues (S-S bond for folding)
- Often glycosylated
What is on the cytoplasmic domain?
CH2 end of peptide
Are the three domains independent?
Yes - interchangeable
What can the ectodomain do?
May circulate as a hormone binding protein
Example of ectodomain circulating as a hormone binding protein
GH receptor ectodomain acts as a GH binding protein in the blood circulation
What happens when the TSH receptor ecrodomain dissociates?
Ectodomain from the TSH receptor may induce antibodies which bind to the receptor and mmic TSH action (cause of hyperthyroidis, in Graves disease)
What is Graves disease?
- Common cause of hyperthydoidism
- An over-production of thyroidism which causes enlargement of the thyroid and other symptoms such as exopthalmos, heat intolerance and anxiety
What does the cytoplasmic domain do?
- Relays the signal to the interior of the cell
- Induces signalling cascade (relay of conformational changes of signalling proteins)
How are conformational changes of signalling proteins induced?
- Hormone binding to receptor --> conformational changes such as:
- Binding between proteins
Many signalling proteins are activated by phosphorylation of which amino acids?
Serine, threonine, tyrosine
Why are Ser, Thr, Tyr amino acids that are phosphorylated?
Have a polar hydroxyl group that is replaced by a phosphate group
In protein phosphorylation, where does the phosphate group come from?
Phosphate donor is ATP
What are the advantages for phosphorylation of proteins as a controlling mechanism for signal transduction?
- Rapid: does not require new protein synthesis or protein degradation
- Reversible: easily reversed by action of protein phosphatases
- Easy to relay signals: phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins
Why is phosphorylation of proteins as a controlling mechanism for signal transduction rapid?
Does not require new protein synthesis or protein degradation
How is phosphorylation of proteins as a controlling mechanism for signal transduction reversible?
Easily reversed by action of protein phosphates
Why is phosphorylation of proteins as a controlling mechanism for signal transduction easy to relay signals?
Phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins
How much of all cellular proteins are phosphorylated?
Which phosphorylated amino acids are more abundant?
Phosphorylated Serines and Threonines are much more abundant tan phosphorylated Tyrosines (100:1)
Why is phosphorylation of tyrosines special?
- Often occurs at the beginning of a signal cascade
- Tyr is bulky
What is the purpose of phosphorylated tyrosines?
Serve as docking sites for down stream signal proteins
What is important about the amino acid sequence that mediates docking to phosphorylated tyrosines?
The amino acid sequence that mediates docking to phosphorylated tyrosines (SH2 and SH3 domains) is conserved and diagnostic for proteins involved in the signalling cascade
What are the three major types of cell surface receptors?
- Intrinsic tyrosine kinase activity
- Recruited tyrosine kinase activity
- G-protein coupled receptor
How do tyrosine kinase coupled receptors work?
- Signal molecule in form of dimer
- Signal molecule binds to TK coupled receptor - makes an active catalytic response
- OR binding recruits a enzyme from outside the receptor and activates
How does G-protein coupled receptor work?
Signal molecule binds and activates G-protein, which binds to enzyme to activate
What is the structure of TK coupled cell surface receptor?
Peptide chain crosses cell membrane ONCE
How does Tyrosine kinase coupled cell surface receptors work?
Have intrinsic tyrosine kinase activity located in the cytosolic domain. The kinase is activated by hormone binding or recruit tyrosine kinase after receptor activation by hormone
What is an example of a receptor with intrinsic TK-kinase activity?
- Insulin receptor
- Most studied
What is the structure of insulin receptors?
- Hetero-tetrameric structure
- 2 α and 2 β chains held together by disulphide bonds
How many insulin receptors per cell, and where are they most abundant?
- Between 100 and 200 000 receptors per cell
- Highest number in adipocytes and hepatocytes
What is the main effect of insulin receptor signalling?
All of the signalling cascades are overlapping and going in different directions but the main effect is taking out the glucose through GLUT4 receptors also stimulating the synthesis of glycogen
What is the sequence of events after insulin binding?
- Autophosphorylation of intracellular domain of receptor
- Docking and phosphorylation of IRS-1 and IRS-2 (insulin receptor substrate)
- Activation of 2 major signal pathways
What does the binding of insulin lead to?
- Change in conformation of receptor complex which activates phosphorylation of tyrosines
- Autophosphorylation of the insulin receptor followed by docking and phosphorylation of insulin-receptor substrates (IRS)
What are the 2 branches of the insulin signal pathway?
- PI 3-kinase pathway
- Gene expression
What does the PI 3-kinase pathway of insulin receptor result in?
- maintenance of active (dephosphorylated) glycogen synthase --> glycogen synthesis increases
- Movement of glucose transporter GLUT4 to the outer membrane --> glucose uptake increases
How does the gene expression branch of insulin pathway work?
- Docking of Grb2 (growth receptor-bound protein) binding to SOS (son of seven less), binding and activation of Ras (replacement of GDP with GTP)
- Phosphorylation cascade: Raf, MEK, to MAPL (mitogen activated kinase)
- MAPK is a kinase central to many cellular responses. Among target proteins are regulatory genes of cell division (c-fos, c-jun)
What is MAPK?
- Kinase central to many cellular responses
- Among target proteins are regulatory genes of cell division (c-fos, c-jun)
What is the net effect of insulin on blood glucose?
- Insulin converts excess blood glucose into 2 storage forms
- 1) glycogen in liver and muscle
- 2) triacylglycerols in adipose tissue
What are some defects in the inulin receptor/signalling pathway?
- Insulin resistance syndromes
- Impaired glucose metabolism, raised insulin concentrations
What is Rabson-Mendenhall syndrome?
Insulin receptor partially functioning
What is Leprechaunish/Donahues syndrome?
Severe intra-uterine growth retardation - most patients dead <1 year
How are receptors with recruited TK activity characterized?
- Characterized by 4 alpha-helices and homology of the ectodomain
- Cytoplasmic domain varies but one region (Box 1) is conserved and appears to be involved in triggering cell division
What are some examples of receptors with recruited TK activity?
Receptors for growth hormone, prolactin and leptin
How many binding sites does GH have?
- GH has 2 binding sites and binds sequentially to two receptor molecules
- Dimerization of the cytoplasmic regions initiates signal transduction
How do GH receptors work?
- GH binds to receptor 1
- GH binds to receptor 2 forming a dimeric complex (receptor 1 and 2 are identical)
- The GH-dimeric receptor complex recruits and activates JAK-2 (tyrosine kinase)
How many pathways are activated by binding of JAK2 to GH receptor?
3 major pathways activated by binding of JAK2 to GHR
What are the three branches of GH signalling pathway?
- 1. Activation of the transcription regulatory proteins STAT (4 different proteins)
- 2. Activation of the MAPK pathway (as insulin, but not dependent on IRS1)
- 3. Activation of PI 3-kinase pathways (as insulin). Responsible for insulin-like metabolic effects of GH
How are JAIKs recruited in the GH binding cascade?
- Binding of interferon cross-links adjacent receptors, and JAKs cross-phosphorylate each other on tyrosines
- Activated JAKs phosphorylate receptors on tyrosines
What is the STATs pathway in the GH binding cascade?
- STAT stands for Signal Transduction and Activation of Transcription
- STAT proteins dock on phosphorylated receptor via SH2 domain and are phosphorylated by JAK proteins
- Dissociation from receptor, dimer formation, migration into the nucleus
- Formation of transcription complex
What is Laron syndrome?
- Defective GH receptor, but normal or elevated levels of GH
- Severely impaired growth
How do G-protein coupled receptors work?
- Bind and activate G-proteins bound to the inner membrane
- Activation of the G-proteins leads to the generation of the 'second' messengers cAMP, DAG, and IP3
- 2nd messenger is depending on the messenger that is activated
What does the second messenger of the GPCR pathway depend on?
The receptor that is activated
What are the three choices for second messenger in the GPCR pathway?
cAMP, DAG, IP3
What are some examples of endocrine hormone receptors that use GPCR?
TRH, GnRH, TSH, FSH, ACTH, GHRH and oxytocin
What is a diagnostic feature of GPCR?
It crosses the membrane SEVEN times
What is formed by the seven transmembrane alpha-helices?
The seven transmembrane alpha-helices form a pore
How is the ectodomain modified?
What does G-proteins use as a switch?
- Use GTP/GDP as a switch
- GTP-bound = G protein on
- GDP-bound = G protein off
- Take GTP off and put GDP on - not necessarily phosphorylation
What happens with the G protein is 'on'?
Leads to activation of adenylate cyclase and/or phospholipase C (in some cases both can be turned on)
What is the subunit structure of G proteins?
- Trimeric structure (alpha, beta, gamma)
- Beta and Gamma bind together to form heterodimer
- Functional units are G-alpha and G-beta/gamma dimer - exist as pool in cell membrane
How is a membrane-bound enzyme (adenylate cyclase) modulated by a GTP protein?
- G-protein is bound to GDP and attached to plasma membrane
- Binding leads to conformation change that allows biding of receptors and subunits
- GTP is added and ATP goes the cAMP while the alpha subunit dissociates
What does Gs-alpha subunit do?
Activates adenylate cyclase
What does Gi-alpha subunit do?
Inhibits adenylate cyclase
What does Gq-alpha subunit do?
Activates phospholipase C
What does Go-alpha subunit do?
Activates ion channels
What is McCune Albright syndrome?
- Gs alpha turned on permanently
- G-signalling pathway is on despite the absence of a hormone stimulant --> skin pigmentation, breast development, precocial puberty associated with ovarian cysts
What is the purpose of cAMP in the GPCR pathway?
- cAMP activates the cAMP dependent kinase (PKA)
- Binding of cAMP to the regulatory protein complex releases active protein kinase A
What is the purpose of PKA in the GCPR pathway?
- PKA phosphorylates the transcription factor CREB (cyclic AMP response element binding protein)
- PKA phosphorylates serines and threonines of the cAMP-response element binding protein (CREB)
- CRB is a transcription factor which switches on genes by binding to the CRE sequence (enhancer sequence) of DNA to induce transcription of cAMP-inducible genes
How is the signal from GPCR terminated?
Signal is terminated by phospho-diesterases which hydrolyse cAMP to 5'AMP (ie regulatory subunits can bind to PKA to activate)
How does the GPCR signal transduction via DAG/Ca++ work?
G protein couples receptor 7 membrane hormone binds conformation
How are the messengers diacylglycerol and Ca2+ generated?
- Activation of membrane associated PLC (3 major isoforms)
- PIP2--> DAG + IP3
- PIP2 is a minor phospholipid of the cell membrane
- DAG and IP3 are second messengers
How do intracellular receptors for hormones work?
- Steroid hormones and thyroid hormones bind to a family of intracellular receptors which are structurally related
- The intracellular receptors are located in the cytoplasm or the nucleus (or both)
- The receptors function as transcription factors (activated by transcription)
- Response is slow since transcription and translation of proteins is necessary
Which kind of hormone produces a slower response and why?
- Steroid and thyroid
- Transcription and translation of proteins is necessary
How does a steroid hormone travel?
- Travels bound to protein so it can travel in an aqueous environment
- Steroid bound to carrier is not biologically active
What are the domains of thyroid and steroid receptors?
- Hormone-specific binding domain (AF2)
- Highly conserved DNA-binding domain
- Transcription activating domain (AF1). Hypervariable. Not dependent on the type of hormone
What are the 2 classes of receptors for steroid and thyroid hormones?
- Class 1: Cytosolic (is not bound to DNA)
- Class 2: Nuclear (already bound to DNA before the ligand binds
How does the DNA-binding domain work?
- Two loops that form a 'zinc finger' (contain zinc atom)
- Class 1 receptors (steroids) form complexes with heat-shock proteins
- Class 1 steroids must bind to activate (remove heat shock protein) then once activated must find DNA to bind to DNA
- Hormone binding dissociates the complex and frees the DMA binding domain
- Class 2 receptors (T3 receptor) are already bound to DNA and are activated by hormone binding
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