1. Chapter 15 – Signal Transduction and G Protein–Coupled Receptors
G protein–coupled receptor (green) bound to β-arrestin (purple).
G protein–coupled receptors activated a prolong period – phosphorylated and bind arrestin, inhibiting further signaling by the receptor.

2. Chapter 15 – Signal Transduction and G Protein–Coupled Receptors
15.1 Signal Transduction: From Extracellular Signal to Cellular Response 15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins 15.3 G Protein–Coupled Receptors: Structure and Mechanism 15.4 G Protein–Coupled Receptors That Regulate Ion Channels 15.5 G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 15.6 G Protein–Coupled Receptors That Trigger Elevations in Cytosolic and Mitochondrial Calcium

3. Signal Transduction and G Protein–Coupled Receptors
15.1 Signal Transduction: From Extracellular Signal to Cellular Response
All cells respond to extracellular signals/stimuli that activate plasma membrane or cytosolic receptors.
Activated receptors function as transcription factors or activate G protein switches that regulate a variety of downstream pathways or induce generation of intracellular second messengers that do so.
Protein phosphorylation by kinases and dephosphorylation by phosphatases regulate protein activity in the cellular pathways and can amplify intracellular signaling.

4. FIGURE 15-1 Overview of cell signaling.
Extracellular signaling molecules – synthesized, packaged into secretory vesicles, and secreted by specialized signaling cells within multicellular organisms
Signal – produces a specific response only in target cells expressing receptor proteins that bind the signal
Hydrophobic signaling molecules – (steroids and related molecules)
Step 1: Diffuse through the plasma membrane
Step 2: Bind to cytosolic receptors
Step 3: Receptor-signal complex moves into the nucleus – binds transcription-control regions in DNA to activate or repress gene expression
Hydrophilic signaling molecules – (small molecules [adrenaline, acetylcholine], peptides [yeast mating factors, glucagon], and proteins [insulin, growth hormone])
Cannot diffuse across the cell membrane
Step 4: Bind to specific cell-surface receptor proteins – triggers receptor conformational change that activates the receptor
Step 5: Activated receptor activates one or more downstream signal transduction proteins or small-molecule second messengers.
Step 6: Signal transduction proteins or small-molecule second messengers activate one or more effector proteins.
Step 7a: Effector – stimulates modification of specific cytosolic proteins; short-term (sec-min) changes in cellular function, metabolism, or movement
Step 7b: Effector – moves into the nucleus; triggers long-term (hours-permanent) changes in gene expression
Termination or down-modulation of the cellular response –
Step 8: Negative feedback/feedback represssion from intracellular signaling molecules
Step 9: Destruction of the extracellular signal

5. FIGURE 15-2 Types of extracellular signaling.
Extracellular molecule signaling – three classifications; based on distance over which the signal acts:
(a) Endocrine: (epinephrine, insulin)
Signaling molecules – synthesized and secreted by signaling cells (e.g., cells in endocrine glands)
Transported through the circulatory system
Affect distant target cells expressing the receptor
(b) Paracrine: (neurotransmitters, growth factors)
Signaling molecules secreted by a cell – affect only nearby target cells expressing the receptor
Some may bind to ECM – released only when ECM is degraded
(c) Autocrine: (growth factors)
Cells respond to signals they secrete. (Tumor cells may overproduce and respond to growth factors.)
(d) Membrane protein signals: signal neighboring cells by direct contact with surface receptors.

6. FIGURE 15-3 Regulation of protein activity by a kinase/phosphatase switch.
Cell-surface receptor signaling – involves kinase phosphorylation and phosphatase dephosphorylation to regulate target protein activity.
Protein kinase – transfers terminal phosphate from ATP to specific Ser/Thr or Tyr–OH (phosphorylated residue is part of a specific kinase target motif)
Protein phosphatase – hydrolyzes P off protein restoring Ser/Thr or Tyr–OH
Protein kinases and phosphatases –
Regulated by signaling processes
Modify specific protein targets containing target motifs
Effect phosphorylation (and reversal by dephosphorylation) on protein activation or deactivation – protein-specific.
Example target protein (reversed in other proteins):
unphosphorylated – inactive
phosphorylated – active

7. FIGURE 15-4 GTPase switch proteins cycle between active and inactive forms.
GTP-binding proteins – signal transduction pathway on-off switches
GTPase protein superfamily (GTP-binding, G protein):
ON/active – GTP bound
OFF to ON –
promoted by GEFs (guanine nucleotide exchange factors)
GEFs catalyze dissociation of bound GDP and replacement by GTP (not phosphorylation of GDP)
OFF/inactive – bound GDP
ON to OFF –
GTPase activity – GTP → GDP + Pi (ON to OFF)
Accelerated by GAPs (GTPase-activating proteins) and RGSs (regulators of G protein signaling)
GTPase switch proteins – two large signaling classes:
Heterotrimeric – activated by direct interaction with surface receptors (GEFs)
Monomeric – activated by GEFs that are activated by surface receptors or other proteins

8. FIGURE 15-5 Switching mechanism of monomeric G proteins.
G protein ON-OFF transition conformational changes:
Involve switch I and switch II (e.g., Ras monomeric G protein)
Promotes binding to downstream signaling proteins
(a) Active/ON state – bound GTP –
Switch I (green) and switch II (blue) – bound to the GTP terminal γ phosphate through interactions with conserved threonine and glycine residues backbone amide groups
Switch domain conformation – can bind and activate specific downstream effector proteins
(b) Inactive/OFF state – bound GDP –
Intrinsic GTPase activity – hydrolyzes GTP to GDP (removes GTP γ phosphate)
GTP hydrolysis rate – time G protein remains in the active conformation
Switch I and switch II relaxation into off conformation – inhibits interaction with downstream effectors
(Similar spring-loaded mechanism switches the alpha subunit in heterotrimeric G proteins between the active and inactive conformations by movement of three switch segments.)

9. FIGURE 15-6 Four common intracellular second messengers.
Intracellular second messengers transmit signals through the cytosol.
cAMP:
Generated from ATP by adenylyl cyclase
Activates PKA
cGMP:
Generated by guanylyl cyclase
Activates PKG and specific cation channels
IP3 and DAG:
Both made from PIP2 by phospholipase C
IP3 – opens channels to release Ca2+ from the ER
DAG – with Ca2+ activates PKC
Calcium ions (Ca2+) (not shown):
Released from intracellular stores or transported into the cell
Activates calmodulin, specific kinases (PKC), and other regulatory proteins

10. FIGURE 15-7 A signal transduction pathway involving a G protein, a second messenger, a protein kinase, and several target proteins.
Generalized signal transduction pathway:
Step 1: Hormone binding to its cell-surface receptor
Step 2: Activated receptor (GEF) activates trimeric G protein
Step 3: G protein alpha subunit binds to and activates second messenger-generating enzyme.
Step 4: Activated enzyme generates multiple second messenger molecules.
Step 5: Second messenger activates a protein kinase.
Step 6: Kinase phosphorylates and changes activity of one or more target proteins.
Step 6a: Cytosolic target proteins induce changes in cellular function, metabolism, or movement.
Step 6b: Target transcription factors induce changes in gene expression.

11. Signal Transduction and G Protein–Coupled Receptors
15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins
Near-maximal response of a cell to a particular ligand generally occurs at ligand concentrations at which less than 100 percent of its receptors are bound to the ligand.
Signal receptors and pathways are targeted by numerous drugs.
Receptors and signaling pathway intermediates are studied with a variety of experimental approaches including affinity chromatography, Western blotting, immunoprecipitation, and pull-down assays.

12. FIGURE 15-8 Binding assays determine the Kd and the number of receptors per cell, but the maximal physiological response to an external signal usually occurs when only a fraction of the receptors are occupied by ligand.
Activation of fewer than 100 percent of receptors usually elicits near-maximal cellular response.
Dissociation constant – measure of receptor affinity for its ligand (Epo-Epo receptor in bone marrow cell)
Experiment to determine the affinity of a receptor for a ligand:
Labeled ligand – added at various concentrations (x axis) to cells that do (experimental cells) and do not (control cells) express the receptor
Incubation in ligand – 1 hr (allows binding) at 4°C (Low temperature prevents endocytosis of the cell-surface receptors.)
Amount of bound ligand (label) is measured (control cell label binding subtracted from binding to receptor-expressing cells).
Plot – bound ligand per cell as a function of the ligand concentration (red)
Results:
At relatively high ligand concentrations – number of receptor-bound ligand molecules approaches number of total cell-surface receptors
Kd (half maximal) ligand binding = 1 nM ligand
Parallel physiological response experiments results (blue):
18 percent receptors bound to ligand – 50 percent of the maximal physiological response
50 percent receptors bound to ligand – near maximal physiological response
Conclusion: relative physiological response is greater than ligand binding.
(Drug binding to ligands used to decrease overactive physiological responses that cause diseases.)

13. FIGURE 15-9 Structures of the natural hormone epinephrine, the synthetic agonist isoproterenol, and the synthetic antagonist alprenolol.
Synthetic analogs of natural hormones are widely used both in research on cell-surface receptors and as drugs – two classes:
May bind much more tightly to the receptor than does the natural hormone
May be more stable
Agonist – mimics function of a natural hormone –
Binds to and activates receptor
Induces the normal cellular response to the hormone
Antagonist – inhibits function of natural hormone
Binds to the receptor ligand-binding site but induces no response
Blocks natural hormone binding
Reduces normal physiological activity of the hormone
Epinephrine receptor drugs:
Structurally similar to natural hormone epinephrine
Isoproterenol – Agonist
Agonist of bronchial smooth muscle cell β2 epinephrine-responsive G protein–coupled receptors
Binds ~10x more strongly (10x lower Kd) than epinephrine
Receptor activation – promotes relaxation of bronchial smooth muscle and opening of lung air passage.
Used in treating bronchial asthma, chronic bronchitis, and emphysema
Alprenolol – Antagonist
Antagonist of cardiac muscle cell epinephrine-responsive G protein–coupled receptor (β1-adrenergic receptor)
Receptor activation increases the heart contraction rate
Antagonists (beta-blockers) slow heart contractions.
Used in treatment of cardiac arrhythmias and angina

14. EXPERIMENTAL FIGURE Activation by the hormone erythropoietin (Epo) of three signal transduction proteins via their phosphorylation.
Western blotting with antibody specific for phosphorylated site reveals signal-dependent phosphorylation of target proteins.
Experiment:
Mouse erythrocyte progenitor cells – treated for 10 minutes with different concentrations of erythropoietin (Epo) hormone. (stimulates differentiation of progenitor cells into red blood cells)
Cell extracts analyzed by Western blotting with antibodies specific for phosphorylated and total (phosphorylated and unphosphorylated) forms of signal transduction proteins in three signaling pathways
Results:
Epo stimulates concentration-dependent phosphorylation of all three signaling proteins – activates all three pathways (Maximal effect at ≥1 unit Epo/ml)
Stat5 – transcription factor phosphorylated on tyrosine 694
Akt – kinase phosphorylated on serine 473
p42/p44 MAP kinase – phosphorylated on threonine 202 and tyrosine 204

15. EXPERIMENTAL FIGURE A pull-down assay shows that the small GTP-binding protein Rac is activated by platelet-derived growth factor (PDGF).
Rac (G protein) – regulates molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form
Pull-down assay – quantifies Rac activation by PDGF
Experiment:
(a) Assay principle: Rac-GTP but not Rac-GDP will bind specifically to the p21-activated protein kinase (PAK1) Rac-binding (PBD) domain.
Cell lysates of hematopoietic stem cells treated for 1 minute (d, 1’) with the hormone platelet-derived growth factor (PDGF) and of untreated control cells (d, 0)
Step 1: Beads with the PAK1 Rac-binding PBD domain attached added to cell lysates – only Rac-GTP will bind to PBD attached to beads
Step 2: Pellet beads to recover attached Rac-GTP
(b) Analysis: Western blot of protein attached to the beads using an anti-Rac antibody (anti-actin used as loading control)
Results: PDGF treatment of cells activates Rac.

16. Signal Transduction and G Protein–Coupled Receptors
15.3 G Protein–Coupled Receptors: Structure and Mechanism
The large diverse family of G protein-coupled receptors (GPCRs) respond to a variety of extracellular signals and activate trimeric G proteins.
G proteins function as On-Off switches for intracellular signaling pathways by activating or inactivating ion channels or effector enzymes that generate second messenger molecules.
GPCR signaling pathways regulate a wide range of cellular activities from metabolism to gene expression.
G Protein–Coupled Receptors: Structure and
Mechanism
• G protein–coupled receptors (GPCRs) are a large and
Diverse family with a common structure of seven membranes spanning
α helices and an internal ligand-binding pocket that
is specific for particular ligands (see Figures and 15-13).
• GPCRs are coupled to heterotrimeric G proteins, which
contain three subunits designated α, β, and γ. The Gα subunit
is a GTPase switch protein that alternates between an
active (“on”) state with bound GTP and inactive (“off”)
state with bound GDP. The “on” form separates from the
β and γ subunits and activates a membrane-bound effector.
The β and γ subunits remain bound together and can also
transduce signals (see Figure 15-14).
• Ligand binding causes a conformational change in certain
membrane-spanning helices and intracellular loops of the
GPCR, allowing it to bind to and function as a guanine nucleotide
exchange factor (GEF) for its coupled Gα subunit, catalyzing
dissociation of GDP and allowing GTP to bind. The
resulting change in the conformation of the switch region in
Gα causes it to dissociate from the Gβγ subunit and the receptor
and interact with an effector protein (see Figure 15-14).
• FRET experiments demonstrate receptor-mediated dissociation
of coupled Gα and Gβγ subunits in live cells (see
Figure 15-15).
• The effector proteins activated (or inactivated) by heterotrimeric
G proteins are either enzymes that form second messengers
(e.g., adenylyl cyclase, phospholipase C) or ion channels
(see Table 15-2). In each case, it is the Gα subunit that determines
the function of the G protein and affords its specificity.
• GPCRs can have a range of cellular effects depending on
the subtype of receptor that binds a ligand. The hormone
epinephrine, for example, which mediates the fight-or-flight
response, binds to multiple subtypes of GPCRs in multiple
cell types, with varying physiological effects.
• Efforts to identify orphan GPCRs led to the discovery of
orexins, hormones that regulate feeding behavior and sleep
in both animals and humans.

17. Table 15-1 Human G Protein–Coupled Receptors of Pharmaceutical Importance
Humans GPCRs – detect and respond to a wide of signals, including neurotransmitters, hormones involved in glycogen and fat metabolism, and photons of light
~30 percent of all drugs used in humans – agonists or antagonists of specific GPCRs or groups of closely related GPCRs
GPCR signal transduction pathways share common elements:
(1) A receptor that contains seven membrane-spanning α helices
(2) A receptor-activated heterotrimeric G protein cycling between GTP-active and GDP-inactive forms
(3) A membrane-bound effector protein
(4) Proteins that desensitize the signaling pathway
Many pathways:
Involve second messengers
Have short-term effects in the cell – rapid activation/inhibition of existing proteins, including enzymes or ion channels

18. FIGURE 15-12 General structure of G protein–coupled receptors.
Human genome – genes for ~800 GPCRs
All G protein–coupled receptors have:
The same orientation in the membrane – N-terminus outside, C-terminus in cytosol
Contain seven transmembrane α-helical regions (H1–H7)
Have four extracellular segments (E1–E4)
Four cytosolic segments (C1–C4)
Ligands have not been identified for many “orphan” receptors.

19. FIGURE 15-13 Binding of ligands to GPCRs.
(a) β1-adrenergic receptor bound to antagonist cyanopindolol (ligand):
H 1-7 – transmembrane helices
E2 – one of 4 extracellular loops
C2, C4 – two of four cytosolic domains
(b) Hormone-binding pocket:
Side chains of 15 amino acids in four transmembrane α helices (3, 5, 6, and 7) and one extracellular loop E2 make noncovalent contacts with the ligand.
Examples of specific binding interactions: N atom in both in cyanopindolol and in epinephrine forms an ionic bond with the carboxylate side chains of helix 3 aspartate 121 (D) and helix 7 asparagine 329 (N).
(c) Glucagon (29-amino-acid peptide) binding to the glucagon receptor:
Glucagon C-terminus (red) – binds to the receptor N-terminal domain
Glucagon N-terminus – thought to insert into a binding pocket that is in the center of the seven transmembrane α helices

20. FIGURE General mechanism of the activation of effector proteins associated with G protein–coupled receptors.
Ligand-activated G protein–coupled receptors – (GEFs) catalyze exchange of GTP for GDP on the α subunit of a heterotrimeric G protein
Box – trimeric G protein Gα and Gβγ subunits – tethered to the membrane by covalently attached lipid molecules (wiggly black lines)
GPCR regulation of effector enzyme activity – (Light color denotes inactive conformation and dark color denotes active conformation of each protein.)
Step 1: Ligand binding induces receptor activation conformational change.
Step 2: Activated receptor binds to trimeric G protein.
Step 3: Activated receptor GEF activity stimulates Gα subunit release of GDP.
Step 4: GTP binding changes Gα conformation –
Dissociates Gβγ (Gβγ subunit activates other effector enzymes in some pathways.)
Activates Gα
Step 5: Gα·GTP activates effector enzyme.
Step 6: Gα intrinsic GTPase activity hydrolyzes GTP to GDP – dissociates Gα and turns off effector enzyme. (G protein active for minutes or less)

21. EXPERIMENTAL FIGURE Activation of a G protein occurs within seconds of ligand binding to its cell-surface G protein–coupled receptor.
Forster resonance energy transfer (FRET) technique:
Wavelength of emitted fluorescence changes when two fluorescent proteins interact.
Reveals kinetics of trimeric G protein activation
Dictyostelium discoideum amoeba signaling pathway: (extracellular cAMP – signaling ligand for G protein–coupled receptor – primary signal, not a second messenger, in this system)
FRET experiment:
Amoeba cells transfected with genes encoding two fusion proteins –
Gα-cyan fluorescent protein (CFP, excited by 440-nm light, fluoresces 490-nm light)
Gβ-yellow fluorescent protein (YFP, excited by 490-nm light emitted from CFP, fluoresces 527-nm light)
(a, left) Inactive Gα∙Gβγ complex – CFP and YFP close enough for energy transfer
Excitation of CFP with 440-nm light –
causes fluorescence energy transfer from activated CFP to YFP
emission of 527-nm (yellow) light instead of 490-nm (cyan) light
(a, right) activated G-protein – dissociation of the Gα and Gβγ subunits – CFP and YFP not close enough for energy transfer
Excitation of CFP with 440-nm light
Loss of 527-nm light emission – emission of 490-nm (cyan) light instead
(b) Results: plot of yellow light (527 nm) emission from a single transfected amoeba cell before and after addition of extracellular cAMP (arrow)
Conclusion: Extracellular signal-GPCR interaction stimulates G protein activation within seconds.

22. FIGURE Structure of the β2-adrenergic receptor in the inactive and active states and with its associated heterotrimeric G protein, Gs.
Ligand binding converts β2-adrenergic receptor into a conformation that binds its trimeric G protein.
(a) (–) Ligand –
β2-adrenergic receptor – no ligand bound – inactive state
Inactive trimeric G protein – Gs: Gαs (dark purple), Gβ (light purple), and Gγ (pink) – unable to interact with inactive receptor.
(b) (+) Ligand –
Ligand binding causes movement of the receptor TM5 and TM6 helices and changes in the C3 loop to create a surface that forms extensive interactions parts of Gαs
Gαs small conformational change –
Lengthening the α5 helix creates a large surface consisting of the N-terminal α-helical segments αN and α5 that bind mainly to receptor TM5 and
TM6.
Triggers release of GDP and Gβγ
(Robert Lefkowitz and Brian Kobilka – awarded 2012 Nobel Prize in Chemistry for work on β2-adrenergic receptor, including structure determination)

23. Table 15-2 Major Classes of Mammalian Heterotrimeric G Porteins and their Effectors
Different G proteins:
Activated by different GPCRs
Regulate different effector proteins
Humans:
21 Gα subunits encoded by 16 genes (several generated by alternative splicing) – specific functions
6 Gβ subunits
12 Gγ subunits
Gβγ subunits – interchangeable activities

24. Signal Transduction and G Protein–Coupled Receptors
15.4 G Protein–Coupled Receptors That Regulate Ion Channels
The cardiac muscarinic acetylcholine GPCR regulates a K+ channel.
Light stimulation of the photosensitive rhodopsin GPCR closes cGMP-gated Na+/Ca2+ channels by regulating a cGMP pathway in retinal cells.
Several mechanisms act to terminate visual signaling.
Adaptation to a wide range of ambient light levels is mediated by movements of the G protein transducin and the inhibitor protein arrestin into and out of the rod-cell outer segment.

25. FIGURE In heart muscle, the muscarinic acetylcholine receptor activates its effector K+ channel via the Gβγ subunit of a Gi protein.
Some G protein–coupled receptors regulate ion channels.
Heart muscle muscarinic acetylcholine receptors activate a G protein that opens K+ channels:
Acetylcholine binds to a muscarinic receptor (also activated by muscarine) receptor
Activated receptor activates a Gαi subunit and its dissociation from the Gβγ subunit.
Gβγ subunit (rather than Gαi-GTP) binds to and opens a K+ channel (effector protein).
Increased K+ exit hyperpolarizes the cardiac muscle cell membrane – reduces heart muscle contraction frequency
Termination (not shown): Gαi hydrolyzes GTP and rebinds to Gβγ.

26. FIGURE Human rod cell.
Rhodopsin GPCR senses light in rod cells.
(a) Rod cell schematic diagram:
Rhodopsin – located in the flattened membrane disks of the cell outer segment
Synaptic body – synapses with one or more interneurons
(b) EM of the region of the rod cell bracketed region in (a) – junction of the inner and outer segments

27. FIGURE 15-19 Vision depends on the light-triggered isomerization of the retinal moiety of rhodopsin.
Activated by absorbing energy from photon of light
Opsin protein lysine 296 amino group – covalently attached to the light-absorbing pigment 11-cis-retinal
Light absorption:
Causes rapid photoisomerization of the bound cis-retinal to the all-trans isomer
Triggers rhodopsin conformational change to the activated unstable intermediate meta-rhodopsin I
Activated rhodopsin activates Gt protein (transducin)
All-trans-retinal –
Quickly dissociates from opsin (within seconds) – unstable covalent linkage cleaves spontaneously
Converted by a series of enzymes in the rod cell and pigmented epithelium back to the cis isome – reattached to opsin

28. FIGURE 15-20 The light-activated rhodopsin pathway and the closing of cation channels in rod cells.
Light activation of rhodopsin – leads to closing of cGMP-gated cation channels
Dark-adapted rod cells:
High level of cGMP – keeps cGMP-gated nonselective cation channels open
Open channels depolarize the plasma membrane to ~−30 mV, considerably more positive (less negative) than resting potential (−60 to −90 mV) typical of neurons and other electrically active cells.
Stimulates neurotransmitter release
Cells exposed to light:
Step 1: Light absorption activates rhodopsin – to R* form
Step 2: R* (GEF) activates Gαt protein to release GDP and bind GTP.
Step 3: Free Gαt·GTP binds to PDE (effector protein, cGMP phosphodiesterase) inhibitory γ subunits
Step 4: Gαt·GTP dissociates inhibitory γ subunits from the PDE catalytic α and β subunits.
Step 5: Activated PDE hydrolyzes cGMP to GMP.
Step 6:
Decrease in cytosolic cGMP concentration
cGMP dissociation from the cation channels closes the channels
Membrane becomes transiently hyperpolarized.
Neurotransmitter release is reduced.
Signal amplification:
Humans – detect a flash of as few as five photons
Each activated rhodopsin can activate 500 Gαt proteins, which activate 250 PDEs
Each PDE (active for fraction of a second) hydrolyzes hundreds of cGMPs.
Total effect – closure of thousands of cation channels causes a significant change in membrane potential
Signal termination: (~0.2 sec; essential for the temporal resolution of vision)
Step 7:
GTP-γ subunit complex binds the RGS9-Gβ5 GTPase-activating (GAP) complex.
GAP stimulates rapid Gαt·GTP hydrolysis to GTP.
Releases PDEγ subunits
PDEγ subunits rebind to α and β subunits to inhibit PDE activity.
cGMP accumulates – opens cation channels
Ca2+-sensing proteins –
Drop in Ca2+ when channels closed
Activate guanylate cyclase – generates cGMP
Rhodopsin phosphorylation and binding of arrestin

29. Figure 15-21 Inhibition of rhodopsin signaling by rhodopsin kinase.
Feedback repression of overactivated rhodopsin
Rhodopsin kinase – phosphorylates light-activated rhodopsin (R*), but not dark-adapted rhodopsin:
Extent of rhodopsin phosphorylation – proportional to the amount of time each rhodopsin spends in the light-activated form
Greater the extent of R* phosphorylation – the greater R* activation of Gαt is reduced
Arrestin binds to the completely phosphorylated rhodopsin – inhibits rhodopsin activation of Gαt
Process of rhodopsin phosphorylation and inactivation by arrestin – completed very quickly, within 50 milliseconds

30. Figure Schematic illustration of transducin and arrestin distribution in dark-adapted and light-adapted rod cells.
Rod cells adapt to varying levels of ambient light by intracellular trafficking of arrestin and transducin.
(a) Dark (vision most sensitive to very low light levels)
Transducin – most localized in outer segment membranes
Arrestin – most localized in inner segment region of the cell
(b) Bright light (vision is relatively insensitive to small changes in light):
Transducin – moved from outer segment to inner segment
Arrestin – moved from inner segment to outer segment
Coordinated movement of transducin and arrestin:
Contributes to ability to perceive images over a 100,000-fold range of ambient light levels
Protein movement mechanism – may involve microtubules and motor proteins

31. Signal Transduction and G Protein–Coupled Receptors
15.5 G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase
GPCRs activate G proteins that activate or inhibit adenylyl cyclase generation of cAMP from ATP and are regulated by feedback repression.
cAMP activates protein kinase A (PKA), which phosphorylates-regulates multiple target proteins including enzymes in cells.
Epinephrine activation of its GPCR in liver and muscle cells stimulates glycogen breakdown into glucose by inhibiting glycogen synthesis and stimulating glycogen breakdown via a kinase cascade.
PKA activation can stimulate gene expression.

32. FIGURE 15-23 Synthesis and hydrolysis of cAMP by adenylyl cyclase and PDE.
>30 different mammalian GPCRs activate Gαs activation of adenylyl cyclase production of cAMP as a second messenger. (Most cell types express one or more such GPCRs.)
Adenylyl cyclase (AC) – catalyzes formation of cyclic cAMP (second messenger) bond from ATP precursor
cAMP phosphodiesterase (PDE) – catalyzes hydrolysis of cyclic bond – AMP (not second messenger)
Similar reactions occur for production and destruction of cGMP second messenger.

33. FIGURE 15-24 Synthesis and degradation of glycogen.
Glycogen breakdown (glycogenolysis):
Occurs in muscle and liver cells in response to epinephrine and glucagon activation of GPCRs
Principal way glucose is made available to all cells needing energy
Glycogen metabolism – two independently regulated enzymes control synthesis and breakdown pathways:
Glycogen synthase: incorporates UDP-glucose into glycogen
Glycogen phosphorylase – releases glucose from glycogen

34. FIGURE 15-25 Hormone-induced activation and inhibition of adenylyl cyclase in adipose cells.
Adenylyl cyclase – stimulated and inhibited by different receptor-ligand complexes
Different ligands binding to multiple receptors:
Lead to activation or inhibition of adenylyl cyclase activity
Presence of both activation and inhibition pathways in same cell – provides fine-tuned control of the cAMP level and downstream cellular responses
Gα·GTP exchange – same mechanism for Gαs (s, stimulatory) and Gαi (I, inhibitory).
Gαs·GTP and Gαi·GTP – interact differently with adenylyl cyclase:
Gαs-coupled receptor ligand binding – activates adenylyl cyclase
Gαi-coupled receptor ligand binding – inhibits the enzyme
Gβγ subunit in stimulatory and inhibitory G proteins – identical

35. FIGURE 15-26 Activation of the catalytic domain of mammalian adenylyl cyclase by binding to Gαs∙GTP.
X-ray crystallographic analysis – pinpointed Gαs·GTP regions that interact with adenylyl cyclase
(a) Mammalian adenylyl cyclase (AC) – membrane-bound enzyme:
Two similar catalytic domains – each convert ATP to cAMP on the cytosolic face of the membrane
Two integral membrane domains – each contains six transmembrane α helices
(b) 3D structure of Gαs·GTP complexed with two fragments of (AC) catalytic domains:
Gαs·GTP α3–β5 loop and the switch II region helix – interact with a specific cytosolic regions of adenylyl cyclase catalytic domains

36. FIGURE 15-27 Structure of PKA and its activation by cAMP.
cAMP diverse effects – mediated through cAMP-dependent protein kinase (PKA) activation – phosphorylates multiple intracellular target proteins expressed in different cell types
cAMP activates protein kinase A by releasing inhibitory subunits.
(a) PKA:
Two catalytic (C) kinase subunits – transfer terminal phosphate from ATP to target protein specific Ser/Thr-OH
Two regulatory (R) subunits
(-) cAMP – bind and inhibit catalytic subunit phosphorylation activity
(+) cAMP – release active catalytic subunits
(b) Regulatory subunit dimer:
Each R subunit –
Two cAMP-binding domains – CNB-A and CNB-B –
cAMP concentration-dependent cooperative cAMP binding – binding of the first cAMP molecule to CNB-B lowers the Kd for binding of the second cAMP to CNB-A
Catalytic subunit binding site (arrow)
Dimerization/docking domain and a flexible linker – binds to an A kinase–associated protein (AKAP)
(c) cAMP binding to the CNB-A domain:
(–) cAMP – CNB-A loop domain (purple) binds to the catalytic (C) subunit
(+) cAMP –
CNB-A glutamate (E200) and arginine (R209) residues – participate in binding of cAMP
cAMP binding causes CNB-A loop conformational change – displaces activate C subunit from the R subunit

37. FIGURE 15-28 Regulation of glycogen metabolism by cAMP and PKA.
Glycogen – synthesis and degradation by different pathways – both regulated by hormone (e.g., epinephrine)-induced activation of PKA
(Active enzymes – darker shades; inactive forms – lighter shades.)
(a) (+) cAMP – activates PKA:
Glycogen synthesis inhibition – PKA phosphorylates-inactivates glycogen synthase (GS)
Glycogen breakdown activation –
PKA phosphorylates-activates glycogen phosphorylase kinase (GPK)
GPK phosphorylates-activates glycogen phosphorylase (kinase cascade)
GP catalyzes first reaction in glycogen breakdown.
PKA phosphorylates-activates a phosphoprotein phosphatase (PP) inhibitor – binding to PP prevents de-phosphorylation of PKA-phosphorylated enzymes
(b) (–) cAMP – inactivates PKA, activates phosphoprotein phosphatase – PP:
Glycogen synthesis stimulation – PP dephosphorylates-activates GS
Glycogen breakdown inhibition – PP dephosphorylates-inactivates GPK

38. Table 15-3 Cellular Responses to Hormone-Induced Rise in cAMP in Various Tissues
cAMP-mediated activation of PKA:
Activated by different GPCRs in different cell types
Activated by multiple GPCRs in some cell types – different receptors activate same Gαs.
Produces different responses in different cell types
PKA:
Phosphorylates different target proteins in different types of cells – activates different pathways
Phosphorylates Ser/Thr within the same sequence motif in different proteins: X-Arg-(Arg/Lys)-X-(Ser/Thr)-Φ (X = any amino acid; Φ = a hydrophobic amino acid)
Epinephrine activation of the fight-or-flight response:
Liver and skeletal muscle – increase in glucose for energy production
Adipose – breakdown of triglycerides for energy production
Cardiac muscle – increased heart rate – O2 and glucose transport to peripheral tissues
Smooth muscle cell relaxation (not shown) – increase blood flow and air intake
Intestine – fluid secretion (use your imagination)

39. FIGURE Amplification of an extracellular signal by a signal transduction pathway involving cAMP and PKA.
Signal transduction pathways amplify the effects of extracellular signals:
Blood levels of epinephrine as low as 10−10 M can stimulate liver glycogenolysis and release of glucose.
GPCRs – low abundance – few hundred or thousand per cell
cAMP signaling – requires 10-6 M or 2 x 106 cAMPs (104x amplification)
Amplification:
Single epinephrine molecule binding to one G protein–coupled receptor – activates up to hundreds of G proteins (amplification)
Each G protein activates 1 AC until G protein hydrolyzes GTP (not amplification).
AC catalyzes the synthesis of a large number of cAMP molecules while activated (amplification).
Two cAMPs activate two PKA catalytic subunits (not amplification).
Activated PKA phosphorylates and activates multiple target proteins (amplification).
Activated target proteins:
Other kinases – kinase cascade (may be multiple steps of amplification; the more steps in such the cascade, the greater the signal amplification)
Enzymes – multiple products (amplification)

40. FIGURE 15-30 Activation of CREB transcription factor following ligand binding to Gαs-coupled GPCRs.
Liver cell PKA activation – also induces the expression of several enzymes involved in gluconeogenesis (conversion of three-carbon compounds such as pyruvate to glucose)
cAMP-PKA regulates gene expression through CREB.
Step 1: Receptor stimulation leads to rise in cAMP.
Step 2: cAMP activates PKA.
Step 3: PKA catalytic subunits translocate into the nucleus.
Step 4: PKA phosphorylates-activates the CREB transcription factor.
Step 5:
Activated CREB forms complex with the co-activator CBP/P300 and other proteins.
CREB complex binds to CRE regulatory elements in promoters of multiple genes.
CREB complex binding stimulates transcription of the various target genes controlled by a CRE.

41. FIGURE Localization of PKA and PDE to the nuclear membrane in heart muscle by an A kinase–associated protein (AKAP).
AKAP family PKA anchoring proteins:
~50 different types
Localize effects of cAMP-PKA to specific regions of the cell
mAKAP:
Anchors both the PKA regulatory subunit (R) and PDE to the nuclear membrane
PKA-PDE negative feedback loop – provides close local control of the cAMP level and PKA activity
1: (–) Hormone – basal PDE activity – maintains cAMP levels below threshold for PKA activation
2: (+) Hormone –
Hormone activation of β-adrenergic receptors increases in local cAMP to a level in excess of that which can be degraded by PDE.
cAMP binding to the PKA R subunits releases the active catalytic (C) subunits into the cytosol.
3: PKA activity
Some C subunits – enter the nucleus and phosphorylate-activate CREB to turn on expression of CRE-genes
Some C subunits – phosphorylate-activate PDE to hydrolyze cAMP back to basal levels – inactivating PKA (negative feedback)
4: PDE dephosphorylation returns the complex to the resting state.
Heart muscle cells (not shown):
AKAP localizes PKA near target plasma membrane Ca2+-channels.
PKA phosphorylation – opens channels
Ca2+ influx through channels – increases contraction
AKAP localization of PKA next to target Ca2+ channels – reduces contraction activation time

42. FIGURE Binding of β-arrestin to phosphorylated GPCRs triggers receptor desensitization and activation of several different signal transduction proteins.
Most GPCRs – down-regulated by feedback repression (an end product of a signaling pathway blocks an early step in that pathway)
Hormonal stimulation for several hours – two mechanisms of feedback repression:
PKA prolonged activation –
phosphorylates several Ser/Thr residues in the receptor cytosolic domain
phosphorylated receptor – binds hormone, but cannot efficiently activate Gαs – decreases adenylyl cyclase activation
(a) BARK (β-adrenergic receptor kinase) activation –
Phosphorylates different receptor Ser/Thr residues
Phosphorylated receptor binds arrestin
Receptor-arrestin complex binds Clathrin-AP2 – endocytosis removes receptor from plasma membrane (some receptors degraded, some dephosphorylated and recycled back to the PM)
β-Arrestin signaling switch – turns off GPCR signaling, turns on other signaling pathways:
transduces signals from activated receptors – activates several cytosolic protein kinases:
Src – activates the MAP kinase pathway – phosphorylation-activation of key transcription factors
JNK-3 (a Jun N-terminal kinase) – phosphorylation-activation of the Jun transcription factor activates expression of stress response proteins
(b) 3-D structure of arrestin binding to rhodopsin domains:
Binds to parts of transmembrane helix 7
Binds to segments of the activated rhodopsin C-terminal cytosolic alpha helix that includes the two phosphorylated resides

43. Signal Transduction and G Protein–Coupled Receptors
15.6 G Protein–Coupled Receptors That Trigger Elevations in Cytosolic and Mitochondrial Calcium
GPCR-G protein activation of phospholipase C generates IP3 (soluble ) and DAG (membrane bound) second messengers from PIP2.
IP3 triggers the opening of IP3-gated Ca2+ channels in the endoplasmic reticulum and elevation of cytosolic free Ca2+, which activates PKC and calmodulin.
Neural and hormonal stimulation coordinately regulate glycogen breakdown through Ca2+ and cAMP.
Acetylcholine activation of its GPCR on endothelial cells induces generation of the NO gaseous signal, which stimulates smooth muscle relaxation and vasodilation.

44. Table 15-4 Cellular Responses to Hormone-Induced Rise in Cytosolic Ca2+ in Various Tissues
G Protein–Coupled Receptors That Trigger Elevations in Cytosolic and Mitochondrial Ca2+.

45. FIGURE 15-33 Synthesis of second messengers DAG and IP3 from phosphatidylinositol (PI).
GPCR-Gαo/Gαq activated phospholipase C generates two key second messengers derived from the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2).
PI-4 kinase and PIP-5 kinase – generate PIP2 precursor from phosphatidyl inositol (a major membrane phospholipid)
Activated phospholipase C hydrolyzes phosphate bond of PI(4,5)P2 – yields membrane-associated DAG and soluble IP3 second messengers
Signal termination:
Phosphatase removes the 5-phosphate from IP3.
A second phosphatase removes the 1-phosphate.
Inositol 4-phosphate is reused to synthesize PI 4-phosphate.

46. FIGURE 15-34 The IP/DAG pathway and the elevation of cytosolic Ca2+.
The IP3/DAG pathway causes a rise in intracellular Ca2+ concentration from ER and external sources from <10-7 to >10-4 M.
(a) Opening of endoplasmic reticulum Ca2+ channels:
Step 1: GPCR activation of either the Gαo or Gαq subunit – activates phospholipase C (PLC).
Step 2: PLC cleaves PI(4,5)P2 – yields IP3 and DAG
Step 3: IP3 diffuses through the cytosol – IP3 interacts with and opens IP3-gated Ca2+ channels in the ER membrane
Step 4: Ca2+ ions move down concentration gradient through the channel into the cytosol.
Step 5: Ca2+ binding activates PKC and its recruitment to the plasma membrane.
Step 6: DAG activates membrane-associated PKC.
Step 7: Activated PKC-Ca2+ leaves membrane to phosphorylate various cellular enzymes and transcription factors, activating proteins involved in cell growth and metabolism.
(b) Opening plasma-membrane Ca2+ channels:
(top) – Low IP3 – High ER Ca2+ levels – Ca2+ ions bind to the transmembrane STIM protein luminal EF hand domains
(bottom) – High IP3 –
Depletion of ER Ca2+ stores dissociates Ca2+ ions from the STIM EF hands.
STIMs oligomerize and relocate to areas of the ER membrane near the plasma membrane.
STIM CAD domains bind to and trigger opening of the plasma membrane Orai1 store-operated Ca2+ channels.
Influx of extracellular Ca2+ contributes to increase in cytosolic Ca2+ concentration.
(c) 3D structure of Orai1 in the closed state:
Composed of six identical subunits arranged around a central Ca2+ pore
Each subunit contains four transmembrane α helices—M1 (blue), M2 (red), M3 (orange), and M4 (violet)—and a helix following M4 that extends into the cytosol (M4 extension helix, violet).
Ca2+ pore – lined by the six M1 helices
Closed state – a Ca2+ ion is bound at the extracellular entrance but cannot enter the pore
Binding of CADs – opens the channel by widening of the pore by the outward movement of the M1 helices, which widens the pore
M1 helices intracellular ends and M4 extensions interact with a portion of the STIM CAD to open the STIM.
The Ca2+-calmodulin complex mediates many cellular responses to external signals (not shown).

47. FIGURE 15-35 Movement of Ca2+ between the cytosol, mitochondrion, and endoplasmic reticulum.
(a) ER:
Main intracellular storage depot for Ca2+
Ca2+-ATPases pump Ca2+ into the ER – establishing Ca2+ cytosolic concentration in unstimulated cell of <10-7 M
Ca2+ movements:
Step 1a: Binding IP3 opens IP3-gated Ca2+ channels (IP3R) in the ER membrane of the endoplasmic reticulum releases Ca2+ into the cytosol.
Step 1b: Binding IP3 opens IP3-gated Ca2+ channels in the ER mitochondria-associated membranes (MAMs).
Step 2: Outer mitochondrial membrane VDACs (voltage-dependent anion channels) –
Physically linked to ER IP3Rs by GRP75 proteins
Linked channels efficiently pass the Ca2+ released from the MAMs into the mitochondria intermembrane space.
Step 3: High concentration of intermembrane space Ca2+ opens MCUs (mitochondrial Ca2+ uniporter).
Ca2+ flows into the mitochondrial matrix – increases ATP synthesis
Step 4: Ca2+ –
Released from matrix into the intermembrane space over time by inner membrane Ca2+/Na+ (NCLX) and Ca2+/H+ (HCX) antiporters to prevent Ca2+ toxicity
VDAC transfers intermembrane space Ca2+ into the cytosol.
Step 5: ER membrane Ca2+-ATPases pump Ca2+ from the cytosol into the ER – restores high ER Ca2+ and low cytosolic Ca2+ levels
(b) Mitochondrial calcium uniporter (MCU) complex:
Multimer of MCU subunits forms the regulated Ca2+ pore.
Additional subunits – include the integral membrane protein EMRE and regulatory subunits MICU1 and MICU2
Ca2+ binding to the MICU subunits opens the MCU pore.
Ca2+ flows through the open MCU pore from the intermembrane space into the matrix.

48. FIGURE Oscillations in the cytosolic Ca2+ concentration following treatment of human HeLa cells with histamine.
Feedback loops trigger spikes in the cytosolic Ca2+ concentration – continuous activation of certain GPCRs induces rapid, repeated spikes in cytosolic Ca2+ concentration
Feedback loop:
Low cytosolic Ca2+ concentration – potentiates IP3 opening of IP3-gated Ca2+ channels
IP3 induced opening of ER IP3-gated Ca2+ channels (and possible subsequent opening of plasma membrane store-operated channels) – rise in cytosolic Ca2+ from <10-7 to >10-4 M Ca2+
High cytosolic Ca2+ level >10-4 M Ca2+:
Decreases Ca2+ channel affinity for IP3 – channels close (Ca2+ – feedback inhibitor of IP3-gated Ca2+ channels)
Inhibits further IP3-induced release of Ca2+ from ER store, even in elevated IP3
Ca2+ ATPase pumps Ca2+ back into ER – lowers cytosolic Ca2+ concentration
Low cytosolic Ca2+ concentration – potentiates IP3 reopening of IP3-gated Ca2+ channels if signal persists
Pituitary gland cells:
Luteinizing hormone–releasing hormone (LHRH) activates GPCR-IP3-dependent Ca2+ release
Ca2+ increase induces secretion of luteinizing hormone (LH).
LH continuous stimulation – feedback loop induces Ca2+ spikes
Each Ca2+ spike induces exocytosis of a few LH-containing vesicles – spikes of LH secretion track Ca2+ spikes

49. FIGURE 15-37 Integrated regulation of glycogenolysis by Ca2+ and cAMP/PKA pathways.
Cells constantly receive multiple signals from their environment.
Integration of Ca2+ and cAMP second messengers regulates glycogenolysis (glycogen breakdown to glucose).
(a) Muscle cells:
Neuronal stimulation –
Cytosolic Ca2+ increase
Ca2+ binds to and activates glycogen phosphorylase kinase (GPK) – increases glycogen breakdown
Epinephrine binding to β-adrenergic receptors –
cAMP increase
cAMP activates PKA
PKA phosphorylates-activates GPK – increases glycogen breakdown
PKA phosphorylates-inhibits glycogen synthase (GS) – inhibits glycogen synthesis
(b) Liver cells:
Hormonal stimulation of two β-adrenergic receptor pathways –
Epinephrine – cAMP increase – activation of PKA
PKA –
Phosphorylates-activates GPK – increases glycogen breakdown
Phosphorylates-inhibits GS – inhibits glycogen synthesis
Vasopressin –
IP3 increase – cytosolic Ca2+ increase
enhances PKC activation by DAG
DAG increase – activation of PKC
PKC – phosphorylates-inhibits GS – inhibits glycogen synthesis

50. FIGURE 15-38 The Ca2+/nitric oxide (NO)/cGMP pathway and the relaxation of vascular smooth muscle.
Signal-induced relaxation of vascular smooth muscle is mediated by a Ca2+-nitric oxide-cGMP-activated protein kinase G pathway.
Endothelial cells signaling to smooth muscle cells:
Acetylcholine activation of its GPCR –
Step 1: G-protein activation of PLC
Step 2: PLC generation of IP3 (+ DAG) – cytosolic Ca2+ increase activates calmodulin
Step 3: Ca2+-calmodulin activates NO synthase
Step 4: NO synthase – generates NO (nitric oxide, gaseous signal molecule)
Step 5: NO diffuses locally into smooth muscle cells – activates guanylyl cyclase (NO receptor)
Step 6: Guanylyl cyclase generates cGMP (second messenger)
Step 7: cGMP activates protein kinase G (PKG).
PKG phosphorylates target proteins – decreases cytosolic Ca2+ – smooth muscle relaxation
Drug intervention:
Nitroglycerin – angina – (chest pain)
Nitroglycerin decomposes to NO – smooth muscle relaxation
Increases blood flow through arteries feeding heart muscle cells
cGMP PDE inhibitor – erectile dysfunction
Prolonged elevation of cGMP in smooth muscle cells
Increased blood flow to penile tissue