Transcript CHAPTER 6

Chapter 32
The Reception and Transmission of
Extracellular Information
Biochemistry
by
Reginald Garrett and Charles Grisham
Garrett and Grisham, Biochemistry, Third Edition
Essential Question
• What are these mechanisms of information
transfer that mediate the molecular basis of
hormone action and that use excitable
membranes to transduce the signals of
neurotransmission and sensory systems?
Garrett and Grisham, Biochemistry, Third Edition
Outline
• What Are Hormones?
• What Are Signal Transduction Pathways?
• How Do Signal-Transducing Receptors
Respond to the Hormonal Message?
• How Are Receptor Signals Transduced?
• How Do Effectors Convert the Signals to
Actions in the Cell?
• What Is the Role of Protein Modules in Signal
Transduction?
Garrett and Grisham, Biochemistry, Third Edition
32.1 – What Are Hormones?
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.1
Nonsteroid hormones
bind exclusively to
plasma membrane
receptors, which
mediate the cellular
responses to the
hormone. Steroid
hormones exert their
effects either by binding
to plasma membrane
receptors or by diffusing
to the nucleus, where
they modulate
transcriptional events.
Classes of Hormones
(There may be others, but we doubt it...)
• Steroid Hormones - derived from cholesterolregulate metabolism, salt/water balances,
inflammation, sexual function
• Amino Acid Derived Hormones - epinephrine,
etc.- regulate smooth muscle , blood pressure,
cardiac rate, lipolysis, glycogenolysis
• Peptide Hormones - regulate many processes
in all tissues - including release of other
hormones
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.2
Structures of some steroid hormones.
Figure 32.3
The conversion of prepro-opiomelanocortin to a family of peptide hormones, including
corticotropin, b-and g-lipotropin, a- and b-MSH, and endorphin.
32.3 – How Do Signal-Transducing
Receptors Respond to the Hormonal
Message?
• Non-steroid hormones bind to plasma
membrane and activate a signaltransduction pathway inside the cell
• Steroid hormones may either
– bind to the plasma membrane
– or
– enter the cell and travel to the nucleus
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Types of Receptors
Three that we know of...
• 7-transmembrane segment receptors
– extracellular site for hormone (ligand)
– intracellular site for GTP-binding protein
• Single-transmembrane segment receptors
– extracellular site for hormone (ligand)
– intracellular catalytic domain - either a
tyrosine kinase or guanylyl cyclase
• Oligomeric ion channels
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7-TMS Receptors
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Receptors that interact with G proteins
Seven putative alpha-helical transmembrane
segments
Extracellular domain interacts with hormone
Intracellular domain interacts with G proteins
Adrenergic receptors are typical
Note desensitization by phosphorylation,
either by bARK or by protein kinase A
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.5
Single TMS Receptors
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Three main classes
Extracellular domain to interact with hormone
Single transmembrane segment
Intracellular domain with enzyme activity
Activity is usually tyrosine kinase or guanylyl
cyclase
Each of these has a "nonreceptor"
counterpart
src gene kinase - pp60v-src was first known
Two posttranslational modifications
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.9
(a) The soluble tyrosine kinase pp60v-src is anchored to the plasma membrane via an Nterminal myristyl group. (b) The structure of protein tyrosine kinase pp60v-src, showing AMPPNP in the active site (ball-and-stick), Tyr416 (red), and Tyr527(yellow). Tyr527 is phosphorylated
(purple).
Figure 32.6
Receptor Tyrosine Kinases
Membrane-associated allosteric enzymes
• How do single-TMS receptors transmit the
signal from outside to inside??
• Oligomeric association is the key!
• Extracellular ligand binding
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.7
Ligand (hormone)-stimulated oligomeric association
of receptor tyrosine kinases.
Guanylyl Cyclases
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Soluble or Membrane-Bound
Membrane-bound GCs are the other group
of single-transmembrane-segment
receptors (besides RTKs)
Peptide hormones activate the membrane
forms
Note speract and resact, from mammalian
ova
Activation may involve oligomerization of
receptors, as for RTKs
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.8
The structure of membrane-bound
guanylyl cyclases.
32.4 – How Are Receptor Signals
Transduced?
Garrett and Grisham, Biochemistry, Third Edition
G Proteins
Many new developments in this area
• Two kinds: "heterotrimeric G proteins" and
"small G proteins"
• X-ray diffraction structures for several of
these are available
• Structures shed new light on possible
functions
Garrett and Grisham, Biochemistry, Third Edition
Heterotrimeric G Proteins
A model for their activity
• Binding of hormone, etc., to receptor
protein in the membrane triggers
dissociation of GDP and binding of GTP to
a-subunit of G protein
• Ga-GTP complex dissociates from Gbg and
migrates to effector sites, activating or
inhibiting
• But it is now clear that Gbg also functions
as a signaling device
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Figure 32.10
Figure 32.11
Second Messengers
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Many and there may be more!
The hormone is the "first messenger"
The second messenger - Ca2+, cAMP or other
- is released when the hormone binds to its
(extracellular) receptor
The second messenger then activates (or
inhibits) processes in the cytoplasm or
nucleus
Degradation and/or clearance of the second
messenger is also (obviously) important
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cAMP and Glycogen
Phosphorylase
Earl Sutherland discovers the first second
messenger
• In the early 1960s, Earl Sutherland
showed that the stimulation of glycogen
phosphorylase by epinephrine involved
cyclic adenosine-3',5'-monophosphate
• He called cAMP a "second messenger"
• cAMP is synthesized by adenylyl cyclase
and degraded by phosphodiesterase
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.12
Cyclic AMP is synthesized by membrane-bound adenylyl cyclase and degraded by
soluble phosphodiesterase.
Figure 32.13
(a) Two views of the complex of the VC1-IIC2 catalytic domain of adenylyl cyclase and Gsa. (b)
Details of the Gsa complex in the same orientation as the structures in (a). SW-1 and SW-2 are
“switch regions,” whose conformations differ greatly depending on whether GTP or GDP is
bound. (Courtesy of Alfred Gilman, University of Texas Southwestern Medical Center.)
How are the hormone receptor
and AC coupled?
• Purified AC and purified receptor, when
recombined, are not coupled.
• Rodbell showed that GTP is required for
hormonal activation of AC
• In 1977, Elliott Ross and Alfred Gilman at
Univ. of Virginia discovered a GTP-binding
protein which restored hormone stimulation to
AC
• Hormone stimulates receptor, which activates
GTP-binding protein, which activates AC
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Signaling Roles for G(bg)
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A partial list
Potassium channel proteins
Phospholipase A2
Yeast mating protein kinase Ste20
Adenylyl cyclase
Phospholipase C
Calcium channels
Receptor kinases
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Stimulatory and Inhibitory G
G proteins may either stimulate or inhibit an
effector.
• In the case of adenylyl cyclase, the
stimulatory G protein is known as Gs and
the inhibitory G protein is known as Gi
• Gi may act either by the Gia subunit
binding to AC or by the Gibg complex
complexing all the Gsa and preventing it
from binding to AC
• Read about the actions of cholera toxin
and pertussis toxin
Garrett and Grisham, Biochemistry, Third Edition
The ras Gene and p21ras
An oncogene and its product
• a gene first found in rat sarcoma virus
• Normal cellular ras protein activates cellular
processes when GTP is bound and is inactive
when GTP has been hydrolyzed to GDP
• Mutant (oncogenic) forms of ras have severely
impaired GTPase activity, so remain active for
long periods, stimulating excessive growth and
metabolic activity - causing tumors to form
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Figure 32.15
The structure of Ras complexed with (a) GDP and (b) GMP-PNP. The Ras
p21-GMP-PNP complex is the active conformation of this protein.
Phospholipases Release Second
Messengers
• Inositol phospholipids yield IP3 and DAG
• PLCb is activated by 7-TMS receptors and
G proteins
• PLCg is activated by receptor tyrosine
kinases (via phosphorylation)
• Note PI metabolic pathways and the role
of lithium
Garrett and Grisham, Biochemistry, Third Edition
Other Lipids as Messengers
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Recent findings - lots more to come
More recently than for PI, other
phospholipids have been found to produce
second messengers!
PC can produce C20s, DAG and/or PA
Sphingomyelin and glycosphingolipids also
produce signals
Ceramide (from SM) is a trigger of
apoptosis - programmed cell death
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.16
(a) The general action of phospholiapase A2 (PLA2), phospholipase C (PLC), and
phospholipase D (PLD). (b) The synthesis of second messengers from phospholipids by the
action of phospholipases and sphingomyelinase.
Figure 32.17
The family of second messengers produced by phosphorylation and breakdown of
phosphatidylinositol. PLC action instigates a bifurcating pathway culminating in two distinct
and independent second messengers: DAG and IP3.
Figure 32.18
Phospholipase C-b is activated specifically by Gq, a GTP-binding protein, and also by Ca2+.
Figure 32.19
Phospholipase C-g is activated by receptor tyrosine kinases and by Ca2+.
Figure 32.20
The amino acid sequences of phospholipase C isozymes b,g, and d share two homologous
domains, denoted X and Y. The sequence g-isozyme contains src homology domains, denoted
SH2 and SH3, SH2 domains (approximately 100 residues in length) interact with
phosphotyrosine-containing proteins (such as RTKs), whereas SH3 domains mediate
interactions with cytoskeletal proteins. (Adapted from Dennis,E.,Three,S., Gi8llah,M., and Hannun,E.,
1991. Role of phospholipases in generating lipid second messengers in signal transduction. The FASEB
Journal 5:2068-2077.)
Ca2+ as a Second Messenger
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Several sources of Ca2+ in cells!
[Ca2+] in cells is normally very low: < 1M
Calcium can enter cell from outside or
from ER and calciosomes
CICR - Calcium-Induced Calcium Release
- is very, very similar to what happens at
the foot structure in muscle cells!
IP3 (made by action of phospholipase C) is
the trigger
See Figures 32.21-32.22
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.21
Cytoplasmic [Ca2+]
increases occur via
the opening of Ca2+
channels in the
membranes of
calciosomes, the
endoplasmic
reticulum, and the
plasma membrane.
Figure 32.22
IP3-mediated
signal transduction
pathways.
Increased [Ca2+]
activates protein
kinases, which
phosphorylate
target proteins.
Ca2+/CaM
represents calcicalmodulin (Ca2+
complexed with
the regulatory
protein
calmodulin).
Calcium Oscillations!
M. Berridge's model of Ca2+ signals
• Ca2+ was once thought to merely rise in
cells to signal and drop when the signal
was over
• Berridge's work demonstrates that Ca2+
levels oscillate in cells!
• The purpose may be to protect cell
components that are sensitive to high
calcium, or perhaps to create waves of
Ca2+ in the cell
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Ca2+-Binding Proteins
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Mediators of Ca2+ effects in cells
Many cellular proteins modulate Ca2+
effects
3 main types: protein kinase Cs, Ca2+modulated proteins and annexins
Kretsinger characterized the structure of
parvalbumin, prototype of Ca2+-modulated
proteins
"EF hand" proteins bind BAA helices
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.23
(a)
Figure 32.24
Helical wheel representations of (a) a model peptide. Ac-WKKLLKLLKKLLKL-CONH2,and (b)
the calmodulin-binding domain of spectrin. Positively charged and polar residues are
indicated in green, and hydrophobic residues are orange. (Adapted from O’Neil,K., and
DeGrado,W., 1990. How calmodulin binds its targets: Sequence independent recognition of amphiphilic ahelices. Trends in Biochemical Sciences. 15:59-64.)
Transduction of two second
messenger signals
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PKC is activated by DAG and Ca2+
Most PKC isozymes have several domains,
including ATP-binding domain, substratebinding domain, Ca-binding domain and a
phorbol ester-binding domain
Phorbol esters are apparent analogues of
DAG
Cellular phosphatases dephosphorylate
target proteins
Read about okadaic acid
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.27
The structure of a phorbol ester. Long-chain fatty acids predominate at the 12-position,
whereas acetate is usually found at the 13-position.
The Polypeptide Hormones
Common features of synthesis
• All secreted polypeptide hormones are
synthesized with a signal sequence (which
directs them to secretory granules, then
out)
• Usually synthesized as inactive
preprohormones ("pre-pro" implies at least
two processing steps)
• Proteolytic processing produces the
prohormone and the hormone
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Proteolytic Processing
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A mostly common pathway
Proteolytic cleavage of the hydrophobic Nterminal signal peptide sequence
Proteolytic cleavage at a site defined by
pairs of basic amino acid residues
Proteolytic cleavage at sites designated by
single Arg residues
Post-translational modification: C-terminal
amidation, N-terminal acetylation,
phosphorylation, glycosylation
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Gastrin as an Example
Heptadecapeptide secreted by the antral mucosa
of stomach
• Gastrin stimulates acid secretion in stomach
• Product of preprogastrin - 101-104 residues
• Signal peptide cleavage leaves progastrin 80-83 residues
• Cleavage at Lys and Arg (basic) residues and
C-terminal amidation leaves gastrin
• N-terminal residue of gastrin is pyroglutamate
• C-terminal amidation involves destruction of Gly
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Protein-Tyrosine Phosphatases
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The enzymes that dephosphorylate Tyr-P
Some PTPases are integral membrane
proteins
But there are also lots of soluble PTPases
Cytoplasmic PTPases have N-term. catalytic
domains and C-terminal regulatory domains
Membrane PTPases all have cytoplasmic
catalytic domain, single transmembrane
segment and an extracellular recognition site
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Soluble Guanylyl Cyclases
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Receptors for Nitric Oxide
NO is a reactive, free-radical that acts either as a
neurotransmitter or as a second messenger
NO relaxes vascular smooth muscle (and is thus
involved in stimulation of penile erection)
NO also stimulates macrophages to kill tumor cells
and bacteria
NO binds to heme of GC, stimulating GC activity
50-fold
Read about NO synthesis and also see box on
Alfred Nobel
Garrett and Grisham, Biochemistry, Third Edition
Protein Modules in Signal
Transduction
• Signal transduction in cell occurs via
protein-protein and protein-lipid
interactions based on protein modules
• Most signaling proteins consist of two or
more modules
• This permits assembly of functional
signaling complexes
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.30
Localization of Signaling Proteins
• Adaptor proteins provide docking sites for
signaling modules at the membrane
• Typical case: IRS-1 (Insulin Receptor
Substrate-1)
– N-terminal PH domain
– PTB domain
– 18 potential tyrosine phosphorylation sites
– PH and PTB direct IRS-1 to receptor
tyrosine kinase - signaling events follow!
Garrett and Grisham, Biochemistry, Third Edition
Signaling Pathways from
Membrane to the Nucleus
• The complete path from membrane to
nucleus is understood for a few cases
• See Figure 32.4
• Signaling pathways are redundant
• Signaling pathways converge and diverge
• This is possible with several signaling
modules on a signaling protein
Garrett and Grisham, Biochemistry, Third Edition
Figure 32.4
A complete signal transduction pathway
that connects a hormone receptor with
transcription events in the nucleus. A
number of similar pathways have been
characterized.
Module Interactions Rule!
• The interplay of multiple modules on many
signaling proteins permits a dazzling array
of signaling interactions
• See Figure 32.30
• We can barely conceive of the probable
extent of this complexity
• For example, it is estimated that there are
approximately 1000 protein kinases in the
typical animal cell - all signals!
Garrett and Grisham, Biochemistry, Third Edition