RECEPTOR

A receptor is a molecule that receives chemical signals from outside a cell. When
such chemical signals bind to a receptor, they cause some form of cellular/tissue
response.

It has two domains

• Ligand binding domain: for binding of a ligand
• Effector domain: for massage propagation
The regulatory actions of a receptor may be exerted directly on its cellular target(s), on
effector protein(s), or may be conveyed by intermediary cellular signaling molecules
called transducers. The receptor, its cellular target, and any intermediary molecules
are referred to as a receptor-effector system or signal transduction pathway.

Second messengers are intracellular signaling molecules released by the cell in

response to exposure to extracellular signaling molecule called the first messengers.
G Protein–Coupled Receptors (GCPRs)
• GPCRs span the plasma membrane as a bundle of seven α-helices.

• Humans express over 800 GPCRs that make up the third largest family of genes in humans, with roughly half of these GPCRs
dedicated to sensory perception (smell, taste, and vision).

Ligands for GPCRs:

1. neurotransmitters such as ACh,

2. biogenic amines such as NE,

3. all eicosanoids and other

4. lipid signaling molecules,

5. peptide hormones,

6. opioids,

7. amino acids such as GABA, and many

8. other peptide and protein ligands.

GPCR classes

• Types: Alpha (α), beta (β)

• Subtypes: α1, α2
• Isoforms: α1A, α1B, α1C,
G proteins

G proteins are signal transducers that convey the information that agonist is bound
to the receptor from the receptor to one or more effector proteins. G–protein-
regulated effectors include enzymes such as adenylyl cyclase, phospholipase C,
cyclic GMP phosphodiesterase (PDE6), and membrane ion channels selective for
Ca2+ and K+. In the basal state of the receptor-heterotrimer complex, the α subunit
contains bound GDP and the α-GDP:βγ complex is bound to the un-liganded
receptor
The α subunits fall into four families (Gs, Gi, Gq, and G12/13) which are
responsible for coupling GPCRs to relatively distinct effectors. The Gs α subunit
uniformly activates adenylyl cyclase; the Gi α subunit can inhibit certain isoforms
of adenylyl cyclase; the Gq α subunit activates all forms of phospholipase C; and
the G12/13 α subunits couple to guanine nucleotide exchange factors (GEFs), such
as p115RhoGEF for the small GTP-binding proteins Rho and Rac. The signaling
specificity of the large number of possible βγ combinations is not yet clear;
nonetheless, it is known that K+ channels, Ca2+ channels, and PI-3 kinase (PI3K)
are some of the effectors of free βγ dimer.
Activation by ligand binding of GPCR
Modulation of effectors
G protein activation

When an agonist binds to a GPCR, there is a conformational change in the receptor

that is transmitted from the ligand-binding pocket to the second and third
intracellular loops of the receptor which couple to the G protein heterotrimer. This
conformational change causes the α subunit to exchange its bound GDP for GTP.
Binding of GTP activates the α subunit and causes it to release both the βγ dimer
and the receptor, and both the GTP-bound α subunit and the βγ heterodimer become
active signaling molecules
Depending on the nature of the α subunit, the active, GTP-bound form binds to and
regulates effectors such as adenylyl cyclase (via Gs α) or phospholipase C (via Gq
α). The βγ subunit can regulate many effectors including ion channels and enzymes
such as PI3-K. The G protein remains active until the GTP bound to the α subunit is
hydrolyzed to GDP. The α subunit has a slow intrinsic rate of GTP hydrolysis that is
modulated by a family of proteins termed regulators of G protein signaling
(RGSs). The RGS proteins greatly accelerate the hydrolysis of GTP and are
potentially attractive drug targets. Once the GDP bound to the α subunit is
hydrolyzed to GDP, the βγ subunit and receptor recombine to form the inactive
receptor-G protein heterotrimer basal complex that can be reactivated by another
ligand-binding event.
Receptor down regulation

Prolonged stimulation of receptor can lead to down regulation of the receptor. This
event is initiated by G protein receptor kinase (GRKs) that phosphorylates the C
terminal tail of the receptor, leading to recruitment of proteins termed arrestin;
arrestin binds to the receptor on internal surface, displacing G proteins and
inhibiting signaling.
SECOND MESSANGERS
Cyclic AMP

Cyclic AMP generated by adenylyl cyclases has three major targets in most cells,
the cyclic AMP dependent protein kinase (PKA), cAMP-regulated guanine
nucleotide exchange factors termed EPACs (exchange factors directly activated by
cAMP), and via PKA phosphorylation, a transcription factor termed CREB (cAMP
response element binding protein).
PKA

The best understood target of cyclic AMP is the PKA holoenzyme consisting of two
catalytic (C) subunits reversibly bound to a regulatory (R) subunit dimer to form a
heterotetramer complex (R2C2). At low concentrations of cAMP, the R subunits
inhibit the C subunits; thus the holoenzyme is inactive. When AC is activated and
cAMP concentrations are increased, four cyclic AMP molecules bind to the R2C2
complex, two to each R subunit, causing a conformational change in the R subunits
that lowers their affinity for the C subunits, causing their activation. The active C
subunits phosphorylate serine and threonine residues on specific protein substrates.
CREB

PKA can phosphorylate a diverse array of physiological targets such as metabolic

enzymes and transport proteins, and numerous regulatory proteins including other
protein kinases, ion channels, and transcription factors. For instance,
phosphorylation of the cAMP response element–binding protein, CREB, on serine
133 recruits CREB-binding protein (CBP), a histone acetyltransferase that interacts
with RNA polymerase II (POLII) and leads to enhanced transcription of ~105 genes
containing the cAMP response element motif (CRE) in their promoter regions (e.g,
angiotensinogen and insulin)
Cyclic AMP–Regulated Guanine Nucleotide Exchange
Factors (GEFs).

The small GTP-binding proteins are monomeric GTPases and key regulators of cell

function. For example, many small GTPases are regulated by GEFs. GEFs act by

binding to the GDP-liganded GTPase and catalyzing the exchange of GDP for GTP.

The two GEFs regulated by cAMP are able to activate members of the Ras small

GTPase family, Rap1 and Rap2; these GEFs are termed exchange proteins activated

by cyclic AMP (EPAC-1 and EPAC-2). The EPAC pathway provides an additional

effector system for cAMP signaling and drug action that can act independently or

cooperatively with PKA.

PKG

Stimulation of receptors that raise intracellular cyclic GMP concentrations leads to

the activation of the cyclic GMP-dependent protein kinase (PKG) that
phosphorylates some of the same substrates as PKA and some that are PKG-
specific. In some tissues, PKG can also be activated by cAMP. Unlike the
heterotetramer (R2C2) structure of the PKA holoenzyme, the catalytic domain and
cyclic nucleotide-binding domains of PKG are expressed as a single polypeptide,
which dimerizes to form the PKG holoenzyme. Pharmacologically important effects
of elevated cyclic GMP include modulation of platelet activation and relaxation of
smooth muscle.
PDEs

PDEs hydrolyze the cyclic 3′,5′-phosphodiester bond in cAMP and cGMP, thereby
terminating their action. The enzymes comprise a superfamily with >50 different PDE
proteins divided into 11 subfamilies. The substrate specificities of the different PDEs
include those specific for cAMP hydrolysis, cGMP hydrolysis, and some that hydrolyze
both cyclic nucleotides. PDE3 inhibitor (milrinone) use in heart failure. PDE5 inhibitors
(e.g., sildenafil) are used in treating chronic obstructive pulmonary disease and erectile
dysfunction. By inhibiting PDE5, these drugs increase accumulation of cellular cGMP in
the smooth muscle of the corpus caverosum, thereby enhancing its relaxation and
improving its capacity for engorgement.
Calcium
Calcium is an important messenger in all cells and can regulate diverse responses
including gene expression, contraction, secretion, metabolism, and electrical
activity. The basal Ca2+ level in cells is maintained by membrane Ca2+ pumps that
extrude Ca2+ to the extracellular space and a sarcoplasmic reticulum (SR) Ca2+-
ATPase (SERCA) in the membrane of the endoplasmic reticulum (ER) that
accumulates Ca2+ into its storage site in the ER/SR.

PLCs are cytosolic enzymes that translocate to the plasma membrane upon receptor
stimulation. When activated, they hydrolyze a minor membrane phospholipid,
phosphatidylinositol-4,5-bisphosphate, to generate two intracellular signals,
inositol-1,4,5-trisphosphate (IP3) and the lipid, diacylglycerol (DAG). Both of these
molecules lead to signaling events by activating families of protein kinases.
DAG directly activates members of the protein kinase C (PKC) family. IP3 diffuses
to the ER where it activates the IP3 receptor in the ER membrane causing release of
stored Ca2+ from the ER. Release of Ca2+ from these intracellular stores raises
Ca2+ levels in the cytoplasm many fold within seconds and activates calmodulin
sensitive enzymes. Depending on the cell’s differentiated function, the
Ca2+/calmodulin kinases and PKC may regulate the bulk of the downstream events
in the activated cells. For example, release of the sympathetic transmitter
norepinephrine onto vascular smooth muscle cells stimulates α adrenergic receptors,
activates the Gq-PLC-IP3 pathway, triggers the release of Ca2+, and leads to
contraction by stimulating the Ca2+/calmodulin-sensitive myosin light chain kinase
to phosphorylate the regulatory subunit of the contractile protein, myosin.
Norepinephrine
Calcium released into the cytoplasm from the ER is rapidly removed by plasma
membrane Ca2+ pumps, and the ER pool of Ca2+ is refilled with extracellular Ca2+
flowing through store-operated Ca2+ channels (SOC) in the plasma membrane.
These currents are termed Ca2+ release-activated currents, or ICRAC. The
mechanism by which ER store depletion opens the store-operated channels requires
two proteins, the channel itself, termed Orai1, and an ER sensor termed STIM1.
Orai1 is highly selective for Ca2+. Under resting conditions, the STIM1 protein is
uniformly distributed on the ER membrane. Release of Ca2+ from the ER stores
results in dimerization of STIM1 and movement to the plasma membrane where
STIM1 and Orai1 form clusters, opening the Ca2+ pore of Orai1 and refilling of the
ER Ca2+ pool.
Transmembrane receptors linked to
intracellular enzymes
Receptor tyrosine kinases
The receptor tyrosine kinases include receptors for hormones such as insulin, for
multiple growth factors such EGF, platelet-derived growth factor (PDGF), nerve
growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth
factor (VEGF), and ephrins. With the exception of the insulin receptor, which has α
and β chains, these molecules consist of single polypeptide chains with large,
cysteine-rich extracellular domains, short transmembrane domains, and an
intracellular region containing one (or in some cases two) protein tyrosine kinase
domains. Activation of growth factor receptors leads to cell survival, cell
proliferation, and differentiation. Activation of the ephrin receptors leads to
neuronal angiogenesis, axonal migration, and guidance.
In addition to recruiting enzymes, phosphotyrosine-presenting proteins can attract
SH2 domain-containing adaptor molecules without activity such as Grb2, which in
turn attract guanine nucleotide exchange factors (GEFs) that can activate the small
GTP-binding protein, Ras. Activation of members of the Ras family leads in turn to
activation of a protein kinase cascade termed the mitogen-activated protein kinase
(MAP kinase or MAPK) pathway. Activation of the MAPK pathway is one of the
major routes used by growth factor receptors to signal to the nucleus and stimulate
cell growth. The first enzyme in the pathway is Rap which is a MAP kinase kinase
kinase (MKKK). Rap phosphorylates and activates a MAP kinase kinase (MKK)
termed MEK. MEK phosphorylates a MAP klnase termed ERK. ERK
phosphorylates a number of transcription factors in the nucleus, including Elk-1 and
CREB, to regulate gene transcription and cause cell proliferation
Spontaneous activating mutations in Ras are responsible for a large fraction of

human cancers; thus, molecules that inhibit Ras are of great interest in cancer

chemotherapy. Drugs that act at receptors in this diverse family include insulin for

the treatment of diabetes mellitus and imatinib, a small molecule protein kinase

inhibitor designed to inhibit both receptor and non-receptor tyrosine kinases.

Imatinib is used to treat chronic myelogenous leukemia and several solid tumors

with dysregulated tyrosine kinases.

JAK-STAT Receptor Pathway.
Cells express a family of receptors for cytokines such as γ-interferon and hormones
like growth hormone and prolactin, which signal to the nucleus by a more direct
manner than the receptor tyrosine kinases. These receptors have no intrinsic
enzymatic activity, rather the intracellular domain binds a separate, intracellular
tryosine kinase termed a Janus kinase (JAK). Upon the dimerization induced by
ligand binding, JAKs phosphorylate other proteins termed signal transducers and
activators of transcription (STATs), which translocate to the nucleus and regulate
transcription.The entire pathway is termed the JAK-STAT pathway. There are four
JAKs and six STATs in mammals which, depending on the cell type and signal,
combine differently to activate gene transcription. For example, prolactin appears to
use JAK1, JAK2, and STAT5 to stimulate milk production.
Receptor Serine-Threonine Kinases.
Protein ligands such as TGF-β activate a family of receptors that are analogous to
the receptor tyrosine kinases except that they have a serine/threonine kinase domain
in the cytoplasmic region of the protein. In the basal state, these proteins exist as
monomers; upon binding an agonist ligand, they dimerize, leading to
phosphorylation of the kinase domain of the type I monomer, which activates the
receptor. The activated receptor then phosphorylates a gene regulatory protein
termed a Smad. There are multiple Smads in cells; once phosphorylated by the
activated receptor on a serine residue, Smad dissociates from the receptor, migrates
to the nucleus, associates with transcription factors and regulates genes leading to
morphogenesis and transformation. There are also inhibitory Smads (the Smad6 or
Smad7 isoforms) that compete with the phosphorylated Smads to terminate
signaling.
Toll-Like Receptors.
Signaling related to the innate immune system is carried out by Toll-like receptors
(TLR). In a single polypeptide chain, these receptors contain a large extracellular
ligand-binding domain, a short membrane-spanning domain, and a cytoplasmic
region termed the TIR domain that lacks intrinsic enzymatic activity. Ligands for
TLR are comprised of a multitude of pathogen products including lipids,
peptidoglycans, lipopeptides, and viruses. Activation of these receptors produces an
inflammatory response to the pathogenic microorganisms. As with all single
membrane-spanning receptors, the first step in activation of TLR by ligands is
dimerization, which in turn causes signaling proteins to bind to the receptor to form
a signaling complex.
Ligand-induced dimerization recruits a series of adaptor proteins including Mal and
the myeloid differentiation protein 88 (MyD88) to the intracellular TIR domain,
which in turn recruit the interleukin-associated kinases termed IRAKs. The IRAKs
autophosphorylate in the complex and subsequently form a more stable complex
with MyD88. The phosphorylation event also recruits TRAF6 to the complex,
which facilitates interaction with a ubiquitin ligase that attaches a polyubiquitin
molecule to TRAF6. This complex can now interact with the protein kinase TAK1
and the adaptor TAB1. TAK1 is a member of the MAP kinase family, which
activates the NF-κB kinases; phosphorylation of the NF-κB transcription factors
causes their translocation to the nucleus and transcriptional activation of a variety of
inflammatory genes.
TNF-α Receptors.

The mechanism of action of tumor necrosis factor α (TNF-α) signaling to the NF-
κB transcription factors is very similar to that used by Toll-like receptors in that the
intracellular domain of the receptor has no enzymatic activity. The TNF-α receptor
is another single membrane-spanning receptor with an extracellular ligand-binding
domain, a transmembrane domain, and a cytoplasmic domain termed the death
domain.
TNF-α binds a complex composed of TNF-receptor1 and TNF-receptor2. Upon
trimerization, the death domains bind the adaptor protein TRADD, which recruits
the receptor interacting protein 1 (RIP1) to form a receptor-adaptor complex at the
membrane. RIP1 is poly-ubiquinated, resulting in recruitment of the TAK1 kinase
and the IκB kinase (IKK) complex to the ubiquinated molecules. The activation
loop of IKK is phosphorylated in the complex eventually resulting in IκBα being
released from the complex allowing the p50/p65 heterodimer of the complex to
translocate to the nucleus and activate the transcription of inflammatory genes.
While there currently are no drugs that interdict the cytoplasmic portions of the
TNF-α signaling pathway, humanized monoclonal antibodies to TNF-α itself, such
as infiximab and adalimumab, are important for the treatment of rheumatoid
arthritis andCrohn’s disease
Receptors That Stimulate Synthesis of cGMP

• Natriuretic Peptide Receptors.

• NO Synthase and Soluble Guanylate Cyclase
Natriuretic Peptide Receptors.
The 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 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. 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). A synthetic BNP agonist, nesiritide, is used for
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).
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+/calmodulin; 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.
Nuclear hormone receptors and
transcription factors
• These receptor proteins are transcription factors able to regulate the expression of
genes controlling numerous physiological processes such as reproduction,
development and metabolism. It comprises of a superfamily of 48 receptors. Well
known members of family include the receptor for steroid hormones such as
androgens, estrogen, glucocorticoid , thyroid hormone and vitamin D. Other
members of family are the receptors for a diverse group of fatty acids, bile acids,
lipid and lipid metabolite. For example; Liver X receptror (LXR), farnesoid X
receptors and peroxisome proliferator-activated receptors (PPAR).
Nuclear hormone receptors contain four major domain in a single polypeptide chain.
The N terminal domain can contain an activation region (AF) essential for
transcriptional regulation followed by a very conserved region with two zinc zingers
that bind to DNA (DNA binding domain). AF region is subject to regulation by
phosphorylation and other mechanisms that stimulate or inhibit the overall ability of
the nuclear receptor to activate the transcription. Hormone response element (HRE)
is a part of DNA specific for binding of particular nuclear receptor. For proper
functioning, a receptor must bind with a ligand, HRE and a co-regulator (to regulate
the target gene).Co-regulator are of following types;

• Co-activator: To increase the gene expression (e.g. CBP/p300)

• Co-repressopr: To suppress the gene expression (e.g. Ncor)

• Co-activators recruit the enzymes to the transcription complex tha modify the
chromatin, such as histone acetylase, which serves to unravel the DNA for
transcription. Co-repressor recruit the protein such as histone deacetylase, which
keeps the DNA tightly packed and inhibits the transcription.

• Tamoxifen (anticancer drug) is an antagonist of estrogen receptor. It recruits the

co-repressor to the transcription factor complex.