Receptors That Stimulate

Synthesis of Cyclic GMP

 Natriuretic Peptide Receptors
 NO Synthase and Soluble Guanylate Cyclase
 Nuclear Hormone Receptors And Transcription
Factors
 The signaling pathways that regulate the synthesis of
cyclic GMP in cells include hormonal regulation of
transmembrane guanylate cyclases such as the atrial
natriuretic peptide receptor (ANP) and the activation of
soluble forms of guanylate cyclase by nitric oxide
(NO).
 The downstream effects of cyclic GMP are carried out
by multiple isoforms of PKG, cyclic GMP-gated ion
channels, and cyclic GMP-modulated
phosphodiesterases that degrade cyclic AM P
(described later).
 Natriuretic Peptide Receptors

 The class of membrane receptors with intrinsic enzymatic

activity includes the receptors for three small peptide ligands
released from cells in cardiac tissues and the vascular system.
 These peptides are atrial natriuretic peptide (ANP), which is
released from atrial storage granules following expansion of
intravascular volume or stimulation with pressor hormones;
brain natriuretic peptide (BNP), which (inspite of its name) is
synthesized and released in large amounts from ventricular tissue
in response to volume overload; and C-type natriuretic peptide
(CNP), which is synthesized in the brain and endothelial cells.
 Like BNP, CNP is not stored in granules;
rather, its synthesis and release are increased
by growth factors and sheer stress on vascular
endothelial cells.
 The major physiological effects of these
hormones are to decrease blood pressure (ANP,
BNP), to reduce cardiac hypertrophy and
fibrosis (BNP), and to stimulate long bone
growth (CNP).
 The transmembrane receptors for ANP, BNP, and CNP
are ligand-activated guanylate cyclases.
 The ANP and BNP receptors contain a ∼450 amino
acid extracellular domain that binds the peptide, a
short 20 amino acid transmembrane domain, and large
intracellular domains that contain a kinase homology
region, a dimerization domain, and a C-terminal
guanylate cyclase domain.
 Phosphorylation of serine residues in the kinase
domain is import ant for activity;
dephosphorylation of these residues leads to
desensitization of the receptor.
 Ligand binding brings the juxta membrane
regions together and stimulates guanylate
soluble forms of guanylate cyclase (GC).
 The cell surface receptors respond to natriuretic peptides
such as atrial natriuretic peptide (ANP) with an increase in
cyclic GMP.
 Soluble guanylate cyclase responds to nitric oxide (NO)
generated from L-arginine by nitric oxide synthase (NOS ).
 Cellular effects of cyclic GMP are carried out by PKG and
cyclic GMP-regulated phosphodiesterases (PDEs).
 In this diagram, NO is produced by a Ca2+/calmodulin-
dependent NOS in an adjacent endothelial cell.
 Detailed descriptions of these signaling pathways are given
throughout the text in relation to the therapeutic actions of drugs
affecting these pathways.
 The ANP receptor (NPR-A) is the molecule that responds
to ANP and BNP .
 The protein is widely expressed and prominent in
kidney, lung , adipose, and cardiac and vascular smooth
muscle cells.
 ANP and BNP play a role in maintaining the normal state of
the cardiovascular system as NPR-A knockout mice have
hypertension and cardiac hypertrophy.
 A synthetic BNP agonist, nesiritide, is used for treatment of
acute decompensated heart failure.
 The NPR-B receptor responds to CNP and has a physical
structure similar to the NPR-A receptor.
 It is also widely expressed but prominent in bone, brain,
kidney, lung , liver, and cardiac and vascular smooth
muscle.
 A role for CNP in bone is suggested by the observation
that NPR-B knockout mice exhibit both dwarfism and
cardiac hypertrophy.
 The natriuretic peptide C receptor (NPR-C) has an
extracellular similar to those of NPR-A and NPR-B but does
not contain the intracellular kinase or guanylate cyclase
domains.
 It has no enzymatic activity and is thought to function as a
clearance receptor, removing excess natriuretic peptide
from the circulation.
NO Synthase and Soluble
Guanylate Cyclase
 Nitric oxide (NO) is a unique signal, a very labile gas
produced locally in cells by the enzyme nitric oxide
synthase (NOS); the resulting NO is able to markedly
stimulate the soluble form of guanylate cyclase to
produce cyclic GMP .
 There are three forms of nitric oxide synthase, neuronal
NOS (nNOS or NOS1), endothelial NOS (eNOS or
NOS3), and inducible NOS (iNOS or NOS2).
 All three forms of this enzyme are widely expressed but
are especially important in the cardiovascular system,
where they are found in myocytes, vascular smooth
muscle cells, endothelial cells, hematopoietic cells, and
platelets.
 NOS produces NO by catalyzing the oxidation of the guanido
nitrogen of L-arginine, producing L-citrulline and NO.
 The enzymes require co-factors including tetrahydrobioptern
and calmodulin.
 The nNOS and eNOS forms of the enzyme are markedly
activated by Ca2+/calmodul in; the inducible form is less
sensitive to Ca2+ but the level of iNOS protein in cells can be
increased over 1000-fold by inflammatory stimuli such as
endotoxin, TNF-α, interleukin-1β and interferon-γ.
 The ability of Ca2+ to activate eNOS and nNOS is important
in certain cells where neurotransmitters that open Ca2+
channels or activate PLC can relax smooth muscle.
 An example is the ability of ACh released by the
parasympathetic nervous system to relax sphincters.
 Soluble guanylate cyclase is a heterodimer composed of α and
β subunits.
 The N-terminal end of the molecule contains a protoporphyrin-
IX heme domain.
 NO binds to this domain at low nM concentrations and
produces a 200- to 400-fold increase in the Vmax of
guanylate cyclase, leading to a marked elevation of cyclic
GMP in the cell.
 The cellular effects of cyclic GMP on the vascular system are
mediated by a number of mechanisms, but especially by PKG.
 For example, in vascular smooth muscle, activation of PKG
leads to vasodilation by:
• Inhibiting IP3-mediated Ca2 release from intracellular stores.
• Phosphorylating voltage-gated Ca2+channels to inhibit
Ca2+influx.
• Phosphorylating phospholamban, a modulator of the
sarcoplasmic Ca2+ pump, leading to a more rapid reuptake of
Ca2+ into intracellular stores.
• Phosphorylating and opening the Ca2+-activated K+channel
leading to hyperpolarization of the cell membrane, which closes
L-type Ca2+channels and reduces the flux of Ca2+ into the cell.
NUCLEAR HORMONE RECEPTORS
AND TRANSCRIPTION FACTORS
 In humans, nuclear hormone receptors comprise a
superfamily of 48 receptors that respond to a diverse set of
ligands.
 The receptor proteins are transcription factors able to regulate
the expression of genes controlling numerous physiological
processes such as reproduction, development, and metabolism.
 Well-known members of the family include receptors for
circulating steroid hormones such as androgens, estrogens,
glucocorticoids, thyroid hormone, and vitamin D.
 Other members of the family are receptors for a diverse group
of fatty acids, bile acids, lipids, and lipid metabolites.
 The latter receptors function as sensors for the metabolic state
of the cell and respond to changes in locally available
molecules.
 Examples include a number of nuclear receptors that are
important in inducing drug metabolizing enzymes, such as the
retinoic acid receptor (RXR); the liver X receptor (LXR—the
ligand is 22-OH cholesterol); the farnesoid X receptor (FXR—
the ligand is chenodeoxycholic acid); and the peroxisome
proliferator-activated receptors (PPARs α, β, and γ 15 deoxy
prostaglandin J2 is one possible ligand for PPARγ the
cholesterol -lowering fibrates bind to and regulate PPARγ).
 In the inactive state, receptors for steroids such as
glucocorticoids reside in the cytoplasm and translocate to the
nucleus up on binding ligand.
 Other members of the family such as the LXR and FXR
receptors reside in the nucleus and are activated by changes in
the concentration of hydrophobic lipid molecules
 Nuclear hormone receptors contain four major domains in a
sing le polypeptide chain.
 The N-terminal domain can contain an activation region (AF-
1) essential for transcriptional regulation followed by a very
conserved region with two zinc fingers that bind to DNA (the
DNA-binding domain).
 The N-terminal activation region (AF-1) is subject to
regulation by phosphorylation and other mechanisms that
stimulate or inhibit the overall ability of the nuclear receptor
to activate transcription.
 The C terminal half of the molecule contains a hinge
region (which can be involved in binding DNA), the
domain responsible for binding the hormone or ligand (the
ligand-binding domain or LBD), and specific sets of amino
acid residues for binding co-activators and co-repressors in a
second activation region (AF-2).
 The x-ray structures of nuclear hormone receptors show that
the LBD is formed from a bundle of 12 helices and that ligand
binding induces a major conformational change in helix 12 .
 This conformational change al so affects the binding of the co-
regulatory proteins essential for activation of the receptor-DNA
complex.
 When bound to DNA, most of the nuclear hormone receptors
act as dimers—some as homodimers, others as heterodimers.
 Steroid hormone receptors such as the glucocorticoid receptor
are commonly homodimers, whereas those for lipids are
heterodimers with the RXR receptor .
 The receptor dimers bind to repetitive DNA sequences, either
direct repeat sequences or an inverted repeat termed hormone
response elements (HRE) that are specific for each type of
receptor (e.g., AGGTCA half-sites oriented as an inverted
repeat with a three-base spacer for the estrogen receptor).
 The hormone response elements in DNA are found up stream
of the regulated genes or in some cases within the regulated
genes.
 An agonist-bound nuclear hormone receptor often activates a
large number of genes to carry out a program of cellular
differentiation or metabolic regulation.
 For example, stimulation of the LXR receptor in hepatocytes
activates 29 genes and inhibits 14 others.
 An important property of these receptors is that they must
bind their ligand, the appropriate HRE, and a co-regulator (from
a family of over 100 proteins coregulators) to regulate their
target genes.
 There are co-activators such as the steroid receptor co-
activator (SRC) family, the p160 family proteins, CARM and
CBP/p 300 or PCG-1α, and co-repressors such as the silencing
mediator of retinoid hormone receptor (SMRT) and nuclear
hormone receptor co-repressor (NCor).
 The activity of the nuclear hormone receptors in a given
cell depends not only on the ligand, but the ratio of co-
activators and co-repressors recruited to the complex .
 Co-activators recruit enzymes to the transcription complex
that modify chromatin, such as histone acetylase, which
serves to unravel DNA for transcription.
 Co-repressors recruit proteins such as histone deacetylase,
which keeps DNA tightly packed and inhibits transcription.
 Depending on the chemical nature of the bound ligand
and the combination of co-activators and co-repressors
recruited to the complex, nuclear hormone receptors may
differentially regulate their target genes.
 This property explains the ability of certain drugs to act as
selective modulators of the receptor and gene expression.
 For example, compounds such as 17β-estradiol are
estrogen receptor agonists in all tissues, whereas
tamoxifen and raloxifene are termed selective estrogen
receptor modulators (SERMs).
 Tamoxifen and raloxifene are partial agonists at the estrogen
receptor; upon binding , these agents elicit unique
conformations of the ligand-binding domain.
 Thus, depending on the specific tissue, different
combinations of co-activators and co-repressors are bound
to the receptor-DNA complex, yielding gene-selective
functions.
 For example, tamoxifen is an antagonist in breast tissue by
virtue of recruiting co-repressors to the transcription factor
complex but is antagonist in the endometrium because it
recruits co-activators.