cAMP Pathway

G-Protein Coupled Receptors


G protein-coupled receptors (GPCRs), also known as seven transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G protein-linked receptors (GPLR), comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. G protein-coupled receptors are found only in eukaryotes, including yeast, plants, choanoflagellates, and animals. The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, and are also the target of around half of all modern medicinal drugs.   G-Protein coupled receptors called so because its ability to bind with trimeric G-proteins (Guanosine nucleotide binding proteins).


Structure of GPCRs:

GPCRs are integral membrane proteins that possess seven membrane-spanning domains or transmembrane helices. The extracellular parts of the receptor can be glycosylated. These extracellular loops also contain two highly-conserved cysteine residues that form disulfide bonds to stabilize the receptor structure.  In 2007, the first structure of a human GPCR was solved.  All receptors of this type have the same orientation in the membrane and contain seven transmembrane -helical regions (H1–H7), four extracellular segments (E1–E4), and four cytosolic segments (C1–C4). The carboxyl-terminal segment (C4), the C3-loop and in some receptors, also the C2 loop are involved in interactions with a coupled trimeric G protein.  G protein-coupled receptors are activated by an external signal in the form of a ligand or other signal mediator. This creates a conformational change in the receptor, causing activation of a G protein. Further effect depends on the type of G protein.


Ligands of GPCRs:


GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, bombesin, bradykinin, endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine, norepinephrine, histamine, glutamate (metabotropic effect), glucagon, acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins, prostanoids, platelet-activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs that act as receptors for stimuli that have not yet been identified are known as orphan receptors.  Whereas, in other types of receptors that have been studied, ligands bind externally to the membrane, the ligands of GPCRs typically bind within the transmembrane domain.


Physiological Roles of GPCRs:


GPCRs are involved in a wide variety of physiological processes. Some examples of their physiological roles include:

  1. The visual sense: the opsins use a photoisomerization reaction to translate electromagnetic radiation into cellular signals. Rhodopsin, for example, uses the conversion of 11-cis-retinal to all-trans-retinal for this purpose
  2. The sense of smell: receptors of the olfactory epithelium bind odorants (olfactory receptors) and pheromones (vomeronasal receptors)
  3. Behavioral and mood regulation: receptors in the mammalian brain bind several different neurotransmitters, including serotonin, dopamine, GABA, and glutamate
  4. Regulation of immune system activity and inflammation: chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system; receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response
  5. Autonomic nervous system transmission: both the sympathetic and parasympathetic nervous systems are regulated by GPCR pathways, responsible for control of many automatic functions of the body such as blood pressure, heart rate, and digestive processes
  6. Cell density sensing: A novel GPCR role in regulating cell density sensing.


Classification of GPCRs:


GPCRs can be grouped into 6 classes based on sequence homology and functional similarity:

!    Class A (or 1) (Rhodopsin-like)

!    Class B (or 2) (Secretin receptor family)

!    Class C (or 3) (Metabotropic glutamate/pheromone)

!    Class D (or 4) (Fungal mating pheromone receptors)

!    Class E (or 5) (Cyclic AMP receptors)

!    Class F (or 6) (Frizzled/Smoothened)


The very large rhodopsin A group has been further subdivided into 19 subgroups (A1-A19). More recently, an alternative classification system called GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin) has been proposed.  The human genome encodes roughly 350 G protein-coupled receptors, which detect hormones, growth factors and other endogenous ligands. Approximately 150 of the GPCRs found in the human genome have unknown functions.


Regulation of GPCRs:


GPCRs become desensitized when exposed to their ligand for a prolonged period of time. There are two recognized forms of desensitization: 1) homologous desensitization, in which the activated GPCR is downregulated; and 2) heterologous desensitization, wherein the activated GPCR causes downregulation of a different GPCR. The key reaction of this downregulation is the phosphorylation of the intracellular (or cytoplasmic) receptor domain by protein kinases.


Phosphorylation by cAMP-dependent protein kinases


Cyclic AMP-dependent protein kinases (protein kinase A) are activated by the signal chain coming from the G protein (that was activated by the receptor) via adenylate cyclase and cyclic AMP (cAMP). In a feedback mechanism, these activated kinases phosphorylate the receptor. The longer the receptor remains active, the more kinases are activated, the more receptors are phosphorylated. In β2-adrenoceptors, this phosphorylation results in the switching of the coupling from the Go class of G-protein to the Gi class. cAMP-dependent PKA mediated phosphorylation is also known as heterologous desensitisation, because it is not specific to ligand bound receptor. In fact any receptor causing an increase in PKA activity will cause increased amounts of this type of desensitisation of other receptors coupled to Go (e.g., dopamine receptor D2 activation may lead to β2-adrenoceptor desensitisation of this type).


Phosphorylation by GRKs


The G protein-coupled receptor kinases (GRKs) are protein kinases that phosphorylate only active GPCRs.  Phosphorylation of the receptor can have two consequences:


Translocation: The receptor is, along with the part of the membrane it is embedded in, brought to the inside of the cell, where it is dephosphorylated within the acidic vesicular environment and then brought back. This mechanism is used to regulate long-term exposure, for example, to a hormone, by allowing resensitisation to follow desensitisation. Alternatively, the receptor may undergo lysozomal degradation, or remain internalised, where it is thought to participate in the initiation of signalling events, the nature of which depend on the internalised vesicle's subcellular localisation.


Arrestin linking: The phosphorylated receptor can be linked to arrestin molecules that prevent it from binding (and activating) G proteins, effectively switching it off for a short period of time. This mechanism is used, for example, with rhodopsin in retina cells to compensate for exposure to bright light. In many cases, arrestin binding to the receptor is a prerequisite for translocation. For example, beta-arrestin bound to β2-adrenoreceptors acts as an adaptor for binding with clathrin, and with the beta-subunit of AP2 (clathrin adaptor molecules); thus the arrestin here acts as a scaffold assembling the componenets needed for clathrin-mediated endocytosis of β2-adrenoreceptors.




G proteins, short for guanine nucleotide-binding proteins, are a family of proteins involved in second messenger cascades.  G proteins are so called because they function as "molecular switches," alternating between an inactive guanosine diphosphate (GDP) and active guanosine triphosphate (GTP) bound state, ultimately going on to regulate downstream cell processes.  G proteins were discovered when Alfred G. Gilman and Martin Rodbell tried to figure out how adrenaline stimulated cells. They found that when a hormone like adrenaline bound to a receptor, the receptor did not stimulate enzymes like adenylate cyclase directly. Instead, the receptor stimulated a G protein, which then stimulated the adenylate cyclase to produce a second messenger, cyclic AMP. For this discovery they won the 1994 Nobel Prize in Physiology or Medicine.  G proteins belong to the larger group of enzymes called GTPases.


Types of G-Proteins:


G protein can refer to two distinct families of proteins. Heterotrimeric G proteins, sometimes referred to as the "large" G proteins that are activated by G protein-coupled receptors and made up of alpha (α), beta (β), and gamma (γ) subunits. There are also "small" G proteins (20-25kDa) that belong to the Ras superfamily of small GTPases. These proteins are homologous to the alpha (α) subunit found in heterotrimers, and are in fact monomeric. However, they also bind GTP and GDP and are involved in signal transduction.


1. Heterotrimeric G-Proteins:


Heterotrimeric G proteins share a common mode of action, i.e., activation in response to a conformation change in the G-protein-coupled receptor, exchange of GTP for GDP and dissociation in order to activate further proteins in the signal transduction pathway. However, the specific mechanism differs between different types of G proteins.  Receptor-activated G proteins are bound to the inside surface of the cell membrane. They consist of the Gα and the tightly associated Gβγ subunits. At the present time, four main families exist for Gα subunits: Gαs, Gαi, Gαq/11, and Gα12/13. These groups differ primarily in effector recognition, but share a similar mechanism of activation.



!    Gαs stimulates the production of cAMP from ATP. This is accomplished by direct stimulation of the membrane-associated enzyme adenylate cyclase. cAMP acts as a second messenger that goes on to interact with and activate protein kinase A (PKA). PKA can then phosphorylate a myriad of downstream targets.

!    Gαi inhibits the production of cAMP from ATP.

!    Gαq/11 stimulates membrane-bound phospholipase C beta, which then cleaves PIP2 (a minor membrane phosphoinositol) into two second messengers, IP3 and diacylglycerol (DAG).

!    Gα12/13 are involved in Rho family GTPase signaling (through RhoGEF superfamily) and control cell cytoskeleton remodeling, thus regulating cell migration.

!    Gβγ sometimes also have active functions, e.g., coupling to L-type calcium channels.


2. Small G-Proteins (Small GTPase):


Small GTPases also bind GTP and GDP and are involved in signal transduction. These proteins are homologous to the alpha (α) subunit found in heterotrimers, but exist as monomers. They are small (20-kDa to 25-kDa) proteins that bind to guanosine triphosphate (GTP). This family of proteins is homologous to Ras GTPases and is also called the Ras superfamily GTPases. 

Function of G-Proteins (G-Protein cycle):


When a ligand activates the G protein-coupled receptor, it induces a conformation change in the receptor (a change in shape) that allows the receptor to function as a guanine nucleotide exchange factor (GEF) that exchanges GTP in place of GDP on the Gα subunit. In the traditional view of heterotrimeric protein activation, this exchange triggers the dissociation of the Gα subunit, bound to GTP, from the Gβγ dimer and the receptor. Both Gα-GTP and Gβγ can then activate different signaling cascades (or second messenger pathways) and effector proteins, while the receptor is able to activate the next G protein.


Once the exchange of nucleotides has occurred, the Ga·GTP complex dissociates from the Gbg subunit, but both remain anchored in the membrane. In most cases, Ga·GTP then interacts with and activates an associated effector protein. This activation is shortlived, however, because GTP bound to G is hydrolyzed to GDP in seconds, catalyzed by a GTPase enzyme that is an intrinsic part of the G subunit. The resulting Ga·GDP quickly reassociates with Gbg    , thus terminating effector activation. In many cases, a protein termed RGS (regulator of G protein signaling) accelerates GTP hydrolysis by the G subunit, reducing the time during which the effector remains activated.  They are also otherwise called as GTPase –activating proteins (GAPs).  The dissociation and association of trimeric subunits of G-Proteins completes one active cycle of G-Protein function and it wan now ready to start new cycle.

Adenylate Cyclase:


Adenylate cyclase (EC, also known as adenylyl cyclase, adenyl cyclase or AC) is a lyase enzyme.  There are ten known adenylate cyclases in mammals: ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9 and ADCY10.




The membrane-bound enzyme contains two similar catalytic domains on the cytosolic face of the membrane and two integral membrane domains, each of which is thought to contain six transmembrane  a helices.

Adenylyl cyclase is a transmembrane protein. It passes through the plasma membrane twelve times.  The important parts for its function are located in the cytoplasm and can be subdivided into the N-terminus, C1a, C1b, C2a and C2b.  The C1 region exists between transmembrane helices six and seven and the C2 region follows transmembrane helix 12. The C1a and C2a domains form a catalytic dimer where ATP binds and is converted to cAMP.


Adenylate cyclase function:


Adenylate cyclase converts 5’-ATP into 3’5’-cyclic AMP and Pyroposhate.

Regulation of Adenylate cyclase:


In adipose cells, ligands like epinephrine, glucagon binding to Gs-coupled receptors causes activation of adenylyl cyclase, whereas ligands like Prostaglandin E and Adenosine binding to Gi-coupled receptors causes inhibition of the enzyme.  The Gbg subunit in both stimulatory and inhibitory G proteins is identical; the Ga subunits and their corresponding recetprs differ.  Ligand-stimulated formation of active Ga.GTP complexes occurs by the same mechanism in both Gs and Gi proteins.  However, Gsa.GTP and Gia.GTP interact differently with adenylyl cyclase, so that one stimulates and the other inhibits its catalytic activity.


5’3’-Cyclic Adenosine Monophosphate (cAMP)


cAMP is an important molecule in eukaryotic signal transduction, a so-called second messenger. Adenylate cyclase can be activated or inhibited by G proteins, which are coupled to membrane receptors and thus can respond to hormonal or other stimuli. Following activation of adenylate cyclase, the resulting cAMP acts as a second messenger by interacting with and regulating other proteins such as protein kinase A and cyclic nucleotide-gated ion channels.  Many different cell responses are mediated by cAMP. These include increase in heart rate, cortisol secretion, and breakdown of glycogen and fat.  If cAMP dependent pathway is not controlled, it can ultimately lead to hyper-proliferation which may contribute to the development and/or progression of cancer.

Molecules that activate cAMP pathway include:


Ø      Cholera toxin - increase cAMP levels

Ø      Forskolin - a diterpine natural product that activates adenylyl cyclase.

Ø      Caffeine and theophylline inhibit cAMP phosphodiesterase which leads to an activation of G proteins that result in the activation of the cAMP pathway

Ø      Bucladesine (dibutyryl cAMP, db cAMP) - also a phosphodiesterase inhibitor

Molecules that inhibit cAMP pathway include:


·        cAMP phosphodiesterase - has an activity opposite to kinase, therefore it dephospohorylates cAMP into AMP, reducing the cAMP levels.

·        Gi protein - inhibitory G protein that inhibits adenylyl cyclase, reducing cAMP levels

·        Pertussis toxin - decrease cAMP level


Regulation of glycogen metabolism by cAMP:


Active enzymes are highlighted in darker shades; inactive forms, in lighter shades.

An increase in cytosolic cAMP activates PKA, which inhibits glycogen synthesis directly and promotes glycogen degradation via a protein kinase cascade. At high cAMP, PKA also phosphorylates an inhibitor of phosphoprotein phosphatase (PP). Binding of the phosphorylated inhibitor to PP prevents this phosphatase from dephosphorylating the activated enzymes in the kinase cascade or the inactive glycogen synthase. 


A decrease in cAMP inactivates PKA, leading to release of the active form of phosphoprotein phosphatase. The action of this enzyme promotes glycogen synthesis and inhibits glycogen degradation.


Degradation of cAMP:


The cAMP level decreased by the action of cyclic nucleotide phosphodiesterase enzyme.  The cyclic nucleotide phosphodiesterases (PDE) comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains.  Intracellular second messengers such as cGMP and cAMP undergo rapid changes in concentration in a response to a wide variety of cell specific stimuli. The concentration of these second messengers is determined to a large extent by the relative synthetic activity of adenylate cyclase and degrative activity of cyclic nucleotide PDE. The role of PDE1 enzymes is to degrate both cGMP and cAMP.


The various isoforms exhibit different affinities for cAMP and cGMP. PDE1A and PDE1B preferentially hydrolyse cGMP, whereas PDE1C degrades both cAMP and cGMP with high affinity. For example in airway smooth muscles of humans and other species, generic PDE1 accounts for more than 50% of the hydrolytic activity of cyclic nucleotides. It has been demonstrated that deletion and overexpression of PDE1 produces strong effects on agonist-induced cAMP signalling but has little effect on the basal cAMP level.



Calmodulin (CaM) has been shown to activate cyclic nucleotide PDE in a calcium-dependent manner and the cooperative binding of four Ca2+ to calmodulin is required to fully activate PDE1. The binding of one Ca2+/CaM complex per monomer to binding sites near the N-terminus stimulates hydrolysis of cyclic nucleotides.  Phosphorylation of PDE1A1 and PDE1A2 by protein kinase A and of PDE1B1 by CaM Kinase II decreases their sensitivity to calmodulin activation. This phosphorylation can be reversed by the phosphatase, calcineurin.


Protein Kinase – A:


In cell biology, protein kinase A refers to a family of enzymes whose activity is dependent on the level of cyclic AMP (cAMP) in the cell. PKA is also known as cAMP-dependent protein kinase (EC Protein kinase A has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism.  Epinephrine and glucagon affect the activity of protein kinase A by changing the levels of cAMP in a cell via the G-protein mechanism, using adenylate cyclase. Protein Kinase A acts to phosphorylate many enzymes important in metabolism. For example, protein kinase A phosphorylates acetyl-CoA carboxylase and pyruvate dehydrogenase. Such allosteric regulation has an inhibitory effect on these enzymes, thus inhibiting lipogenesis and promoting net gluconeogenesis. Insulin, on the other hand, decreases the level of phosphorylation of these enzymes, which instead promotes lipogenesis. 


PKA helps transfer/translate the dopamine signal into nerve cells. It has been found (postmortem) to be elevated in the brains of smokers, in the nucleus accumbens, which mediates reward and motivation: a part of the brain acted on by "virtually all" recreational drugs, as well as "in the area of the midbrain that responds to dopamine, which acts as a 'reward chemical' in smokers and former-smokers."


Activation of PK-A:


Each PKA is a holoenzyme that consists of two regulatory and two catalytic subunits. Under low levels of cAMP, the holoenzyme remains intact and is catalytically inactive. When the concentration of cAMP rises, cAMP binds to the two binding sites on the regulatory subunits, which leads to the release of the catalytic subunits.  The free catalytic subunits can then catalyse the transfer of ATP terminal phosphates to protein substrates at serine, or threonine residues. This phosphorylation usually results in a change in activity of the substrate. Since PKAs are present in a variety of cells and act on different substrates, PKA and cAMP regulation are involved in many different pathways.



In protein synthesis PKA first directly activates CREB, which binds the cAMP response element, altering the transcription and therefore the synthesis of the protein. This mechanism generally takes longer time (hours to days).

Inactivation of PK-A:


PKA is thus controlled by cAMP. Also, the catalytic subunit itself can be regulated by phosphorylation.  Downregulation of protein kinase A occurs by a feedback mechanism: One of the substrates that are activated by the kinase is a phosphodiesterase, which quickly converts cAMP to AMP, thus reducing the amount of cAMP that can activate protein kinase A.


Regulation of Protein kinase-A by PDE and cAMP:


This A kinase–associated protein mAKAP anchors both PKA and cAMP phosphodiesterase (PDE) to the nuclear membrane, maintaining them in a negative feedback loop that provides close local control of the cAMP level.


Step 1: The basal level of PDE activity in the absence of hormone (resting state) keeps cAMP levels below those necessary for PKA activation.

Step 2: Activation of b-adrenergic receptors causes an increase in cAMP level in excess of that which can be degraded by PDE. The resulting binding of cAMP to the regulatory (R) subunits of PKA releases the active catalytic (C) subunits.

Step 3: Subsequent phosphorylation of PDE by PKA stimulates its catalytic activity, thereby driving cAMP levels back to basal and causing reformation of the inactive PKA. Step 4: Subsequent dephosphorylation of PDE returns the complex to the resting state.


Role of Protein Kinase-A in Glycogen Metabolism:


Cyclic AMP Response Element Binding (CREB) Protein:


CREB (cAMP response element binding) is a protein that is a transcription factor. It binds to certain DNA sequences called cAMP response elements (CRE) and thereby increases or decreases the transcription, and thus the expression, of certain genes.  Genes whose transcription is regulated by CREB include: c-fos, the neurotrophin BDNF (Brain-derived neurotrophic factor), tyrosine hydroxylase, and many neuropeptides (such as somatostatin, enkephalin, and corticotropin releasing hormone).  CREB is closely related in structure and function to CREM (cAMP response element modulator) and ATF-1 (activating transcription factor-1) proteins. CREB proteins are expressed in many animals, including humans.


Mechanism of Action:


Receptor stimulation leads to the activation of adenylate cyclase with the help of G-proteins.  Activated adenylate cyclase forms cAMP which inturn activates protein kinase A.  Activated PK-A then translocated to nucleus through nucleus pore.  These activated PK-A, phosphorylates the transcription factor CREB.  Phoshorylated CREB then binds with CBP/P300 (co-activator) and forms activator for cyclic AMP response element.  This activator then binds with CRE and express various genes and proteins.   The DNA binding of CREB is mediated via its basic leucine zipper domain (bZIP domain).

Function of CREB:


CREB has many functions in many different organs although most of its functions have been studied in relation to the brain. CREB proteins in neurons are thought to be involved in the formation of long-term memories; this has been shown in the marine snail Aplysia, the fruit fly Drosophila melanogaster, and in rats. They are necessary for the late stage of long term potentiation. There are activator and repressor forms of CREB. Flies genetically engineered to overexpress the inactive form of CREB lose their ability to retain long term memory. CREB is also important for the survival of neurons, as shown in genetically engineered mice, where CREB and CREM were deleted in the brain. If CREB is lost in the whole developing mouse embryo, the mice die immediately after birth, again highlighting the critical role of CREB in promoting survival.  Disturbance of CREB function in brain can contribute to the development and progression of Huntington's Disease. Abnormalities of a protein which interacts with the KID domain of CREB, the CREB binding protein (CBP) is associated with Rubinstein-Taybi syndrome. CREB is also thought to be involved in the growth of some types of cancer.