Download (LB) domain

Part I => CARBS and LIPIDS
§1.6 Signal Transduction
§1.6a Endocrine Hormones
§1.6b Hormone Receptors
Section 1.6a:
Endocrine Hormones
Synopsis 1.6a
- Hormones are chemical messengers that play a key role in cellular signaling—they
differ from growth factors and cytokines in that they are exclusively produced by
endocrine glands and travel in the blood to act at distant sites (systemic action)
- On the other hand, growth factors and cytokines are produced throughout the
body by numerous cell types and they can act both locally and systemically
- Be aware that the terms hormones, growth factors and cytokines are often used
interchangeably as there is no rigid boundary separating their classification and
their overlapping functions only add to the confusion
- The effect of endocrine hormones (as well as growth factors and cytokines) is
almost always mediated via a cascade of signaling proteins and receptors, one or
more of which are usually enzymes
- While there is a diverse array of endocrine hormones, they can be broadly divided
into two major categories on the basis of their chemical structure:
(1) Micromolecular hormones—these are small molecules such as the
steroid hormones
(2) Macromolecular hormones—these are peptides and proteins such as
insulin and glucagon
Endocrine System: Endocrine Glands
- Glands are specialized organs in the body that synthesize
and secrete chemical products such as sweat and insulin
- Glands are divided into two major categories—exocrine
and endocrine
- Exocrine glands secrete their products via ducts to
specific internal or external sites—examples are
sweat glands, salivary glands and mammary glands
- Endocrine (ductless) glands secrete their products directly
into the bloodstream—examples include pituitary gland,
thyroid gland, adrenal glands, ovaries and testes
- Not all glands are monolithic—eg pancreas acts both as an
endocrine and exocrine gland
- The islets of Langerhans (2%) of pancreas act as an
endocrine gland and secrete metabolic hormones such as
insulin and glucagon directly into the bloodstream
- However, the bulk (98%) of pancreas is an exocrine gland
that secretes via the pancreatic duct a cocktail of digestive
enzymes such as trypsin and chymotrypsin into the small
intestine
Endocrine System: Endocrine Signaling
- In response to specific stimuli, the ductless endocrine glands secrete hormones directly into the
bloodstream so that they can be carried to a distant site (target cells and tissues)
- Upon arrival at their target site, endocrine hormones elicit specific biological effects such as the
maintenance of homeostasis (homeo  “same”; stasis  “static/steady”)—a steady-state in
which living cells tend to maintain a relatively stable and constant environment via regulation of
factors such as temperature, glucose, salt and water balance
Endocrine System: Micromolecular Hormones
Cortisol
(Adrenal glands)
Aldosterone
(Adrenal glands)
Testosterone
(Testes)
Estradiol
(Ovaries)
Progesterone
(Ovaries)
- Micromolecular hormones such as steroid hormones regulate numerous physiological functions central to homeostasis
- Steroid hormones (highly hydrophobic/lipophilic) exert their effects by virtue of their ability to diffuse through the membrane
and binding to their specific intracellular (cytoplasmic and nuclear) receptors called steroid hormone receptors (SHRs)
- SHRs are a subfamily of nuclear receptor superfamily—a group of transcription factors that become activated upon the
binding of a ligand such as a hormone or a vitamin
- Steroid hormones are subdivided into five major classes according to their physiological functions:
Class
Example
Receptor
Major stimuli
Principal Function
Glucocorticoids
Cortisol
Glucocorticoid receptor
Stress, hypoglycemia
Metabolism and inflammation
Mineralocorticoids Aldosterone
Mineralocorticoid receptor Hypotension, acidosis
Osmoregulation—salt and
water balance
Androgens*
Testosterone
Androgen receptor
Exercise, being stress-free Male sex steroid
Estrogens*
Estradiol
Estrogen receptor
Exercise, being stress-free Female sex steroid
Progesterones*
Progesterone
Progesterone receptor
Exercise, being stress-free Menstruation, pregnancy &
embryogenesis
*Produced in both males and females but in reciprocal quantities!
Endocrine System: Macromolecular Hormones
(29aa)
Glucagon
(PDBID 1GCN)
- Macromolecular hormones such as insulin and glucagon—secreted by the
islets of Langerhans of pancreas—regulate blood sugar level
β-chain
(30aa)
- Secreted in response to high blood glucose level, insulin aids the absorption of
glucose from the bloodstream by muscle and adipose cells as well as inhibiting
the production of glucose by the liver—insulin exerts such effect by virtue of its
ability to bind and activate the cell surface insulin receptor—a member of the
receptor tyrosine kinase (RTK) family
S-S
α-chain
(21aa)
S-S
Insulin
(PDBID 4NIB)
- Secreted in response to low blood glucose level, glucagon does the opposite of insulin—it
stimulates the liver to release glucose through the breakdown of glycogen (glycogenolysis) and the
synthesis of glucose from non-carbohydrate precursors such as pyruvate and lactate
(gluconeogenesis)—glucagon exerts such effect by virtue of its ability to bind and activate the cell
surface glucagon receptor—a member of the G-protein-coupled receptor (GPCR) family
- Upon their activation, insulin and glucagon receptors set off a cascade of downstream events within
the cytoplasm to transduce the insulin or glucagon message into the desired response
- From a homeostatic perspective, insulin and glucagon act antagonistically to maintain a relatively
constant blood glucose level (~1mg/ml)—irrespective of external food intake—in non-diabetics
Exercise 1.6a
-
Explain why only certain cells respond to hormones even
though all cells in the body are exposed to the hormone
-
List hormones produced by the pancreas and adrenal
glands. What types of molecules are these hormones?
-
Summarize the biological effects of insulin and glucagon
-
Summarize the biological effects of steroid hormones
Section 1.6b:
Hormone Receptors
Synopsis 1.6b
- How do endocrine hormones drive cellular processes as diverse as cell growth
and cell proliferation through metabolism to the development and progression
of many cancers? Enter hormone receptors!
- Hormone receptors mediate extracellular signals in the form of hormones at the
cell surface (or after their diffusion into the cytosol) to downstream targets such
as transcription factors in the nucleus via what are termed “signaling cascades”
- Hormone receptors and many proteins involved in coupling them to
downstream cellular targets are “modular”—ie they are functionally subdivided
into semi-autonomous regions called “modules” or “domains”
- Among a wide plethora of protein modules, Src homology domains such as SH2
and SH3 play a central role in directly coupling activated hormone receptors to
downstream targets
- Hormone receptors can be broadly divided into three major categories:
(1) Steroid hormone receptors (SHRs)
(2) Receptor tyrosine kinases (RTKs)
(3) G-Protein-coupled receptors (GPCRs)
(1) SHRs: Structural Organization
N
TA
DB
LB
C
- Steroid hormone receptors (SHRs)—a subfamily of nuclear receptor superfamily (~50
members in humans)—are water-soluble intracellular (cytoplasmic and nuclear) proteins that
act as ligand-modulated transcription factors—ie they require a hormone for activation
- SHRs are generally comprised of the TA-DB-LB modular architecture (though alternative but
highly illogical nomenclatures also float around in the literature):
DNA-Binding (DB) domain
Binds to promoters of target genes in a sequence-dependent manner—but only upon
ligand binding to the LB domain!
Ligand-Binding (LB) domain
Recruits other cellular proteins such as transcription factors, co-activators and co-repressors
to gene promoters in a ligand-dependent manner—hormone binding to the LB domain is a
pre-requisite for such functional output
Transactivation (TA) domain
Synergizes the action of LB domain by recruiting additional cellular proteins required for the
assembly of fully functional transcriptional machinery at the target gene promoters—unlike
the LB domain, the TA domain acts in a ligand-independent manner but its role is essential
(1) SHRs: A Typical Signaling cascade
- SHRs usually exist as monomers in
complex with heat shock proteins
(HSPs) in the cytoplasm
Steroid
Hormone
- After diffusion through the cell
membrane, the binding of the
hormone to the LB domain
results in its dimerization
- Dimeric SHR translocates to the
nucleus and binds to the target
gene promoters via its DB domain
LB
LB
LB LB
+
TA
- Recruitment of cellular factors required
to assemble the transcriptional
machinery at the gene promoters is
aided by the LB and TA domains
- This turns on gene expression of
specific proteins—which in turn set
about causing changes to the cell in
response to the hormone
DB DB
DB
DB
HSPs
SHR
TA
TA
TA
Nucleus
LB LB
mRNA
DB DB
DNA
TA
TA
Transcriptional
machinery
(2) RTKs: Structural Organization
Extracellular
Lipid
Bilayer
LB
LB
LB
ADP
ATP
TM
TM
Cytoplasmic
TK
TK
LB
Ligand
Binding
Autophosphorylation
& Dimerization
TM TM
P
TK TK
P
- Receptor tyrosine kinases (RTKs) are single-transmembrane cell surface receptors comprised of:
(1) Extracellular ligand binding (LB) domain
(2) Single α-helical transmembrane (TM) domain
(3) Cytoplasmic tyrosine kinase (TK) domain
- Upon the binding of a cognate ligand (such as a hormone, cytokine or growth factor) to extracellular
LB domain, the receptors either dimerize or undergo a conformational change so as to bring their
cytoplasmic TK domains close together—such proximity and orientation allows each TK domain to
phosphorylate its dimeric counterpart @ a specific Tyr residue in a trans-fashion—ie each TK
phosphorylates the other and vice versa!
- Autophosphorylation of RTKs in such a manner results in their activation, thereby allowing them to
recruit specific signaling proteins to the site of inner membrane surface—this in turn sets off a
cascade of downstream events ultimately culminating in the nucleus
(2) RTKs: Autophosphorylation
ADP + Pi
H2O + ATP
Kinase
Phosphatase
- Protein phosphorylation @ one of the hydroxy amino acids (eg Tyr, Thr or Ser) within
protein chains is by far the most ubiquitous form of post-translational modification
(PTM) observed in proteins—why so?
- Protein phosphorylation essentially serves as a “molecular switch” in that it can turn
protein function “ON” or “OFF”—courtesy of protein kinases and phosphatases
working in tandem to maintain cellular homeostasis
- Such molecular switches are a hallmark of the living machinery, and particularly, the
cellular signaling pathways that need to be tightly regulated in a highly spatial and
temporal manner—ie with respect to both the location of the signaling event (spatial)
and the short time that it should be turned on or off (temporal/transient)—for the
failure to do so often forms the basis of the development and progression of disease
- The fact that the regulation of RTKs is also under the control of phosphorylation offers
a fitting tribute to the importance of kinases and phosphatases to the vitality of life at
molecular level—the human genome encodes ~500 protein kinases and ~200 protein
phosphatases!
(2) RTKs: Family Members
- RTKs are one of the two subfamilies of the larger protein tyrosine kinase (PTK)
superfamily comprised of close to 100 unique members—the other being the nonreceptor tyrosine kinases (nRTKs), or simply the cytoplasmic tyrosine kinases
- Prominent members of the RTK family include:
RTK
Synonyms
IR
EGFR
Protein/Peptide Ligand(s)
Principal Function
Insulin (I), insulin-like growth factors (IGFs) Glucose homeostasis
ErbB1/HER1 Epidermal growth factor (EGF)
Cell growth and proliferation
PDGFR
Platelet-derived growth factor (PDGF)
Cell growth and differentiation
FGFR
Fibroblast growth factor (FGF)
Cell growth and development
VEGFR
Vascular endothelial growth factor (VEGF) Development of blood vessels
HGFR
Met
Hepatocyte growth factor (HGF)
Embryonic development
NGFR
TrkA
Nerve growth factor (NGF)
Neuronal development
EphR
Ephs
Ephrin (Eph)
Cell adhesion and migration
(2) RTKs: Insulin receptor
- Insulin receptor (IR)–involved in the regulation of glucose homeostasis—binds insulin and
insulin-like growth factors such as IGF1 and IGF2
- IR is unusual in that it exists in a disulfide-linked dimeric state even in the unbound
conformation (α and β chains are covalently linked together via disulfide bridges)—ligand
binding merely causes a conformational change that promotes its autophosphorylation
and subsequent activation
(2) RTKs: A Typical Signaling Cascade
- Ligand binding to RTK induces receptor
dimerization and/or autophosphorylation
Ligand
- Activated RTK serves as a binding site for
the recruitment of adaptors such as GRB2 (via its P
SH2 domain) to the inner membrane surface
P
(IMS) in a phosphorylation(Tyr)-dependent manner
Extracellular
Ras
RTK
GDP
GTP
P
P
- Since GRB2 adaptor exists in complex with SOS exchange
factor, the recruitment of the guanine nucleotide exchange
factor SOS to the IMS catalyzes GDP-GTP exchange in Ras,
thereby resulting in its activation
- Next, activated Ras binds and activates Raf kinase
SH3
SH2
Raf
SOS
GRB2 SH3
MEK
MAPK
Jun
P
P
Cytoplasm
Nucleus
MAPK
- Raf kinase then activates the kinase MEK via Ser/Thr
phosphorylation
- This is followed by the activation of the MAP kinases (MAPKs)
such as ERK2 by MEK, also via Ser/Thr phosphorylation
Ras
P
mRNA
P
Jun
- Activated MAPK translocates to the nucleus and phosphorylates specific transcription factors (eg
Jun/Fos/Myc)
- Phosphorylated Jun binds to its promoter within the target genes and turns on gene expression of
specific proteins—which in turn set about causing changes to the cell in response to the ligand
(3) GPCRs: Structural Organization
- G-protein-coupled receptors (GPCRs) are seventransmembrane cell surface receptors with close
to 1000 members—they not only transduce
hormone signals but also ligands/stimuli as diverse
as light (eg rhodopsin), odors, pheromones and
neurotransmitters
- In the absence of a ligand, the cytoplasmic tail of
GPCRs binds to the GDP-bound α-subunit of the so
called “G-proteins”—which are membraneanchored heterotrimers of α, β and γ subunits—
such union between GPCRs and G-proteins locks
the latter in an inactive state
- Upon ligand binding (eg glucagon) at their
extracellular face, GPCRs undergo a
conformational change that results in GDP-GTP
exchange within the α-subunit, thereby allowing it
to dissociate off both the GPCR cytoplasmic tail
and its heterotrimeric partners β and γ subunits
- Such dissociation enables both the GTP-bound αsubunit and the β/γ-heterodimer to act as
“modulators” of other membrane-bound proteins
such as adenylate cyclase (AC) and phospholipase
C (PLC)
GPCR
Ligand (L)
“Inactive”
G-protein (αβγ)
L
GDP-GTP
exchange
“Active”
(3) GPCRs: Adenylate Cyclase (AC)
Adenosine
triphosphate (ATP)
3’-5’-Cyclic adenosine
monophosphate (cAMP)
- Upon activation by the GTP-bound α-subunit of G-proteins, the integral membrane
protein AC catalyzes the conversion of ATP to cAMP
- cAMP then acts as an intracellular “secondary messenger” in the cytosol in response
to extracellular hormone (primary messenger) signal acting through the GPCRs
- Within the cytosol, cAMP targets and modulates the activities of other cellular
proteins such as protein kinase A (PKA)—a kinase involved in the regulation of
carbohydrate and lipid metabolism by virtue of its ability to phosphorylate Ser/Thr
residues in target proteins so as to effect gene expression
(3) GPCRs: Phospholipase C (PLC)
- Upon activation by the GTP-bound α-subunit of G-proteins, the cytoplasmic peripheral
membrane protein PLC catalyzes the hydrolysis of PIP2 (a membrane phospholipid) @ its
glycero-phosphoester bond into IP3 and DAG
- IP3 and DAG then act as intracellular “secondary messengers” in the cytosol in response to
extracellular hormone (primary messenger) signal acting through the GPCRs
- Within the cytosol, IP3 triggers the opening of Ca2+ channels in the endoplasmic reticulum—
the sudden increase in cytosolic concentration of Ca2+ together with DAG leads to activation
of proteins such as protein kinase C (PKC)—a kinase involved in modulating numerous
signaling cascades by virtue of its ability to phosphorylate Ser/Thr residues in target
proteins so as to effect gene expression
Exercise 1.6b
- How does a receptor tyrosine kinase phosphorylate itself?
- Summarize various members of RTK family, their ligands and
functions
- Summarize the roles of proteins such as Grb2, SOS, Ras, and various
protein kinases involved in coupling activated RTKs to downstream
cellular targets such as transcription factors in the nucleus
- Explain why cells contain an array of protein phosphatases as well as
protein kinases
- Summarize the steps of signal transduction from a GPCR to
phosphorylation of target proteins by PKA
- What is the function of a secondary messenger such as cAMP?
Bottled Water Exposed—a Convenience or Health Risk!
Polyethylene
Terephthalate
(PET)
- Plastic water bottles are typically made from a polymer called polyethylene terephthalate (PET)
in combination with a myriad of other chemicals and metals such as antimony (Sb)
- During storage, transportation and exposure to heat, these nasty chemicals directly (or after
decomposition) leach into the water as carcinogens and can often act as endocrine disruptors
- Endocrine disruptors mimic the action of endocrine hormones such as estrogen, thereby
disrupting signaling pathways, promoting cancer and other cellular defects—eg several lines of
evidence suggest that sustained exposure to antimony (a metalloestrogen) contributes to the
development of breast cancer
- Not only tap water is FREE but it is better of the two evils—but avoid water filters as they also
contain nasty carcinogenic compounds (anything that is made from plastic is malastic to health!)