Metabolic Endocrinology - Hormone Action

• 2 ways:
• Pathways with cell-surface receptors
• Pathways with intracellular receptors

• 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

• As long as hormone is bound to carrier protein it is soluble
• Small hydrophobic signal molecule
• Enter through lipid membrane

• Ectodomain - NH2 and glycosylations
• Hydrophobic transmembrane domain - 25 AA needed to cross memebrane
• Cytoplasmic domain

• Ectodomain - NH2 end of peptide
• Rich in cystei residues (S-S bond for folding)
• Often glycosylated

CH2 end of peptide

Yes - interchangeable

May circulate as a hormone binding protein

GH receptor ectodomain acts as a GH binding protein in the blood circulation

Ectodomain from the TSH receptor may induce antibodies which bind to the receptor and mmic TSH action (cause of hyperthyroidis, in 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

• Relays the signal to the interior of the cell
• Induces signalling cascade (relay of conformational changes of signalling proteins)

• Hormone binding to receptor --> conformational changes such as:
• Phosphorylation
• Binding between proteins

Serine, threonine, tyrosine

Have a polar hydroxyl group that is replaced by a phosphate group

Phosphate donor is ATP

• 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

Does not require new protein synthesis or protein degradation

Easily reversed by action of protein phosphates

Phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins

10%

Phosphorylated Serines and Threonines are much more abundant tan phosphorylated Tyrosines (100:1)

• Often occurs at the beginning of a signal cascade
• Tyr is bulky

Serve as docking sites for down stream signal proteins

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

• Intrinsic tyrosine kinase activity
• Recruited tyrosine kinase activity
• G-protein coupled receptor

• 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

Signal molecule binds and activates G-protein, which binds to enzyme to activate

Peptide chain crosses cell membrane ONCE

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

• Insulin receptor
• Most studied

• Hetero-tetrameric structure
• 2 α and 2 β chains held together by disulphide bonds

• Between 100 and 200 000 receptors per cell
• Highest number in adipocytes and hepatocytes

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

• Autophosphorylation of intracellular domain of receptor
• Docking and phosphorylation of IRS-1 and IRS-2 (insulin receptor substrate)
• Activation of 2 major signal pathways

• 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)

• PI 3-kinase pathway
• Gene expression

• maintenance of active (dephosphorylated) glycogen synthase --> glycogen synthesis increases
• Movement of glucose transporter GLUT4 to the outer membrane  --> glucose uptake increases

• 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)

• Kinase central to many cellular responses
• Among target proteins are regulatory genes of cell division (c-fos, c-jun)

• Insulin converts excess blood glucose into 2 storage forms
• 1) glycogen in liver and muscle
• 2) triacylglycerols in adipose tissue

• Insulin resistance syndromes
• Impaired glucose metabolism, raised insulin concentrations

Insulin receptor partially functioning

Severe intra-uterine growth retardation - most patients dead <1 year

• 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

Receptors for growth hormone, prolactin and leptin

• GH has 2 binding sites and binds sequentially to two receptor molecules
• Dimerization of the cytoplasmic regions initiates signal transduction

• 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)

3 major pathways activated by binding of JAK2 to GHR

• 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

• Binding of interferon cross-links adjacent receptors, and JAKs cross-phosphorylate each other on tyrosines
• Activated JAKs phosphorylate receptors on tyrosines

• 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

• Dwarfism
• Defective GH receptor, but normal or elevated levels of GH
• Severely impaired growth

• 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

The receptor that is activated

cAMP, DAG, IP3

TRH, GnRH, TSH, FSH, ACTH, GHRH and oxytocin

It crosses the membrane SEVEN times

The seven transmembrane alpha-helices form a pore

Glycosylation

• 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

Leads to activation of adenylate cyclase and/or phospholipase C (in some cases both can be turned on)

• 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

• 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

Activates adenylate cyclase

Inhibits adenylate cyclase

Activates phospholipase C

Activates ion channels

• 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

• cAMP activates the cAMP dependent kinase (PKA)
• Binding of cAMP to the regulatory protein complex releases active protein kinase A

• 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

Signal is terminated by phospho-diesterases which hydrolyse cAMP to 5'AMP (ie regulatory subunits can bind to PKA to activate)

G protein couples receptor 7 membrane hormone binds conformation

• 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

• 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

• Steroid and thyroid
• Hydrophobic
• Transcription and translation of proteins is necessary

• Travels bound to protein so it can travel in an aqueous environment
• Steroid bound to carrier is not biologically active

• Hormone-specific binding domain (AF2)
• Highly conserved DNA-binding domain
• Transcription activating domain (AF1). Hypervariable. Not dependent on the type of hormone

• Class 1: Cytosolic (is not bound to DNA)
• Class 2: Nuclear (already bound to DNA before the ligand binds

• 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