September 10, 2001.
 
1. 	Transport of hormones
2. 	General characteristics of receptors
3. 	Regulation of receptor binding
4. 	The effects of receptor activation
-  Plasma membrane surface receptors
-  Intracellular receptors
5.  The elimination of the signal

transport of hormones to the site of action

Circulation:  Fee hormone + Bound hormone (carrier proteins, cytosol of RBC) = Total hormone

Carrier proteins:

•	produced by liver
•	synthesis is regulated (e.g., testosterone binding protein in pregnancy)
•	provide a circulating pool for the hormone
•	limit the availability of the hormone on the target site
•	prolong the half-life of hormone
•	certain cells have receptors to certain carrier-hormone complexes
•	bind lipid soluble hormones (But also:  GH-BP, CRH-BP)

Local and systemic circulation

•	Venous blood from GI tract is collected by hepatic portal circulation  GI hormones and hormones given orally delivered to liver before entering the inferior vena cava  first-passage phenomenon

•	the importance of specialized local circulations:  a hormone produced by a cell population reaches the target cells before entering the systemic circulation  target cells exposed to very high concentrations of the hormone.  E.g.,

-	Pituitary portal circulation (hypothalamus  pituitary)
-	Adrenal circulation (cortex  medulla)
-	Circulation of the pancreatic islets (central  peripheral)

Transport through barriers

•	endocrine tissues: fenestrated capillaries  most target tissues: nonfenestrated

•	special barriers (tight junctions, double layer of epithelium or other cell types):

-	blood-brain barrier (except:  circumventricular organs)
-	blood-testis barrier
-	placental barrier

mechanisms of hormone action

receptor concept

•	Hormones exert their biologic action upon binding to specific receptors.
•	Only cells with specific receptors respond to specific hormones (ligands).
•	Specificity of receptors:  one type of receptor binds one specific hormone (or may also bind other, structurally closely related hormones)
•	A target cell displays receptors for various hormones 
•	A certain receptor type may be present in 100 – 1,000,000 copies on a single cell.
•	Hormone-receptor binding activates intracellular effector mechanisms

Receptor types:	1.  Plasma membrane surface receptors:  peptide hormones, catecholamines, melatonin
2.  Intracellular (cytosolic or nuclear) receptors:  steroid and thyroid hormones

Characteristics of receptor binding:
1. 	Binding capacity of receptors
2. 	Receptor occupancy:  low receptor occupancy may be sufficient to evoke maximal response  (e.g., 2% of insulin receptor occupancy evokes maximal response in adipocytes:  due to the presence of spare receptors)
3. 	Affinity between receptor and ligand:  determines if a hormone binds or not to the receptor
Kd (affinity constant):  the hormone concentration at which 50% of the receptors occupied by he hormone (the higher the affinity the lower the Kd)

4.  Intrinsic activity:  determines the intensity of the intracellular response after binding

Receptor ligands:  1.  Agonists
2.  Antagonists

Receptor binding vs.  Non-specific binding

high affinity, low capacity vs.  low affinity, high capacity binding

Regulation of receptor binding

1.	Homologous receptor regulation:  a hormone affects the characteristics of its own receptors 
2.	Heterologous receptor regulation:  a hormone affects the characteristics of other hormones’ receptors
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Change in receptor number:  1.  Receptor upregulation
2.  Receptor downregulation (internalization of the receptors by endocytosis or suppressed receptor synthesis)

Change in receptor affinity:  1.  Positive cooperativity
2.  Negative cooperativity

The effects of receptor activation

1.	Direct effects on membrane ion channels (surface receptors)
2.	Direct activation of intracellular enzymes (surface receptors)
3.	Activation of G proteins/second messenger system (surface receptors)
4.	Receptor internalization (surface receptors)
5.	Direct activation of specific genes (intracellular receptors)

Altered structure of preexisting protein
or
Synthesis of a new protein

I.  plasma membrane surface receptors

Structure:  Transmembrane proteins (glycoproteins)
-  Extracellular moiety (ligand binding site):  N-terminal
-  Membrane-spanning segment(s) (single pass transmembrane protein, multipass t.p.)
-  Intracellular moiety (catalytic site)

Life cycle of receptors:
-	receptor synthesis begins in rER  Golgi apparatus  mature receptors inserted into the plasma membrane, available for ligand binding
-	receptor-hormone complex internalized  endosomes fuse with lysosomes and hormone degraded
-	receptors recycled, ~50 cycles or more before receptor undergoes degradation

A.  Receptors directly associated with ion channels or intracellular enzymes

1.   ion channel‑linked receptors

Cell membrane is impermeable to the majority of ions:  ion movement through transmembrane protein “tunnels” (ion channels)

Ion distribution is different on the two sides of membrane, e.g.,
-	EC Na+  > IC Na+  
-	IC K+ > EC K+
-	IC protein–  >> EC protein–  

Electropotential difference:
IC:  negative
EC:  positive
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Hormone binding to ion channels

Conformational change

Closing or opening ion channels

Ion flux

Membrane potential changes:  excitatory or inhibitory

E.g., 	certain ACh receptor subtypes
certain glutamate receptor subtypes
GABA receptors
serotonin receptors

2.  enzyme-linked receptors

Intracellular moiety itself has enzymatic activity or is associated directly with intracellular enzymes ® the enzyme produces intra-cellular messengers (called “second messengers”).

E.g., 	Receptor tyrosine kinase (e.g., insulin receptor)
Guanylyl cyclase membrane receptors (e.g., ANP receptor)

B.  G protein-linked receptors

The receptor modulates ion channels or intracellular enzymes by activating special membrane proteins (G proteins)

Receptor:   	single polypeptide chain with a seven-pass transmembrane domain;  the most common type of receptors

G proteins:  act as “middlemen” between receptor and second messenger.
GTP-GDP binding activity
3 subunits:  , , and 

When receptors are  not activated, G proteins are present in inactive form:  3 subunits form one complex
 unit binds GDP (GDP---γ complex)

In response to ligand binding to the receptor (i.e., receptor activation):  
GDP- is phosphorylated (= the  subunit is activated), GTP--- complex is formed

GTP- dissociates from complex and moves to an effector membrane protein (ion channel or enzyme)

GTP- binds to the effector protein

GTP- alters the conformation of the effector protein

the function of the effector protein is changed (stimulated or inhibited)  second messenger system is activated (or inhibited)  IC concentration of second messengers is changed

-GTP dephosphorylated to -GDP

-GDP moves back to form GDP--- complex 

The activation of the  subunit continues in a cyclic fashion as long as the receptor is activated by its ligand.

second messengers

1.  cyclic AMP

G protein activation ( cholera toxin permanently activates G protein)

adenylyl cyclase activation

cAMP

cAMP-dependent protein kinase (PKA) activation

The phosphorylation of specific intracellular target proteins 

e.g., cAMP responsive element binding protein (CREB) phosphorylated

CREB binds to CRE site of the regulatory region of genes

Transcription

New protein synthesis

Brakes in the system:  phosphodiesterase
phosphoprotein phosphatase

2.  cyclic gmp 

Guanylyl cyclase
cGMP-dependent protein kinase (PKG)

3.  inositoltriphosphate/diacylglycerol

G protein activation

Phospholipase C activation

Phosphatidylinositol biphosphate (PIP2)  inositoltriphosphate (IP3) + diacylglycerol (DAG)

IP3  diffuses throughout the cytoplasm and mobilizes Ca++ from IC calcium stores (sER)

cytosolic Ca++ 

Ca++ binds to calcium-binding proteins, e.g., calmodulin 

Ca++/calmodulin-dependent protein kinases (CaM-kinases) activated

Specific intracellular target proteins are phosphorylated

the activity of specific intracellular enzymes changes

DAG   remains anchored to the membrane 

activates protein kinase C in the membrane

PKC acts on ion channels and/or activates transcription factors (e.g., nuclear factor-B, MAP-kinase

4.  Arachidonic acid derivatives

G protein activation
¯
Phospholipase A2 activation
¯
Phosphatidylinositol hydrolyzed into arachidonic acid

	5-lipoxygenase	cyclooxygenase

	leukotriens	prostaglandins

II.  intracellular hormone receptors

Ligands: steroid and thyroid hormones, nitric oxide, carbon monoxide

Location:  cytosolic or intranuclear

Types:	-  steroid hormone receptor superfamily (steroid and thyroid h.)
-  arylhydrocarbon receptor (endogenous ligand is not known; environmental toxins (xenobiotics))
-  soluble guanylyl cyclase (NO and CO)		

Structure of steroid hormone receptors:
  	-  hormone binding site (responsible for hormone specificity)
-  DNA binding site (responsible for gene specificity)

In the absence of specific ligand (hormone):  
	
a multiprotein complex is formed:  receptor + molecular chaperone machinery

chaperone peptides = heat shock proteins (Hsp59, Hsp70, Hsp90)	

Hormone binding site is exposed, DNA binding site is covered by chaperone peptides ® the receptor cannot bind to DNA but  ready to bind a ligand

After ligand (hormone) binding:  chaperone proteins dissociate from receptor
								
					DNA binding site of the receptor is free 
								
					receptor + hormone complex binds to the “hormone response element”
					(HRE) sites of specific genes, e.g., 
- estrogen-response element
- progesterone-response element
- thyroid hormone-response element
- glucocorticoid-response element
- Vitamin D-response element
								
					specific genes are turned on
								
							mRNA
								
						new protein is synthesized

Integration of multiple signals by the cell

Cells are simultaneously exposed to a number of hormones

distinct hormones acting on distinct receptors may affect the same cellular function

hormones may act as functional agonists or functional antagonists

Additive effect:  	combined effect of two functional agonists equals the sum of their separate effects
Synergistic effect:  	combined effect of two functional agonists exceeds the additive effects (e.g., FSH and testosterone effects on spermatogenesis)
Permissiveness:   	a hormone has no detectable effect by itself but it has to be present for the full action of another hormone, e.g., thyroid hormones  the number of epinephrine receptors

the elimination of the signal

The short-lived nature of the signal is essential for the adaptation of the minute-to-minute needs of the organism

Half-life:  the time during which the hormone’s concentration is decreased to 50% of the initial concentration

Mechanisms:

-	Degradation of the hormone by the target cell (cellular internalization)
-	Degradation of the hormone by specific organs (liver, kidney, lung)
-	Degradation of the hormone in the circulation
-	Excretion of the hormone