Signal Transduction Pathway - The cAMP Pathway

cAMP stands for cyclic-adenosine monophosphate and we all know that this is the secondary messenger. The concept as to why cAMP is the secondary messenger was first discovered by Earl Sutherland in 1958, when he discovered that the action of hormone, epinephrine, was mediated by an increase in the concentrations of cAMP. Why it is the secondary messenger? Because the first messenger is the hormone itself (here, epinephrine is the primary messenger) and the cAMP mediates the activity of primary messenger.

How is cAMP formed and then degraded? So, cAMP is formed by the action of an enzyme, adenylyl cyclase which acts on ATP (adenosine triphosphate) as can be seen in the above diagram. This cAMP is further degraded to AMP by cAMP phosphodiesterase.

cAMP has an important function that it mediates the breakdown of glycogen to glucose in muscle cells for energy. But how? This effect and some more effects of cAMP in turn is mediated by action of another enzyme, known as protein kinase A (abbreviated as PKA), also called as cAMP dependent protein kinase. Now, next question is how is PKA regulated? To understand this, first we will have to understand the structure of PKA. The enzyme PKA has four subunits. Two of the subunits are regulatory while the other two are catalytic (figure on the right side). Now, when cAMP binds to regulatory subunits, there is conformational change which leads to the dissociation of the catalytic subunits. These free catalytic subunits are enzymatically active and they phosphorylate the serine residues on the target molecules (proteins).


Now, in effect of glycogen metabolism, how does PKA regulates glycogen metabolism?
The PKA has two enzymes upon which it acts (as can be seen in the adjacent diagram). The first one is another protein kinase called, phosphorylase kinase. This kinase is phosphorylated on serine residue by PKA; which then activates glycogen phosphorylase. The glycogen phosphorylase is an enzyme in glycogenolysis or glycogen breakdown pathway and thus is responsible for breakdown of glycogen to glucose-1-phosphate. Another enzyme which PKA phosphorylates is glycogen synthase. The glycogen synthase is the enzyme which is responsible for glycogen synthesis. The point to note here is that the phosphorylation of glycogen synthase inhibits its enzymatic activity. Hence, from here, we can infer that, the increase in cAMP levels results in the activation of PKA and this stimulates glycogen breakdown and at the same time, also inhibits glycogen synthesis.

As already mentioned, there are many other effects of cAMP. Lets have a look at another example. When there is an increase in cAMP levels, there is activation of transcription of various target genes in many animal cells. These target genes are known to contain the specific regulatory sequence called as the cAMP response element, generally abbreviated as CRE (diagramatic representation on the right side). Again, as described above, when cAMP binds to regulatory subunit of PKA, the catalytic subunit is released which carries the signal from cytoplasm to nucleus. Within the nucleus this activated PKA phosphorylates a transcription factor called CREB (CRE binding protein) at serine residue. This in turn recruits various co-activators and transcription of cAMP inducible genes takes place. This regulation of gene expression plays an important role in various processes like proliferation, differentiation, survival etc.

It is important to note that protein phosphorylation needs to be balanced which is done by the activity of protein phosphatases. Some protein phosphatases are transmembrane receptors while others are cytosolic. These phosphatases remove the phosphate group from tyrosine or serine or threonine residues in the substrate proteins thereby terminating the responses which is initiated by the activity of protein kinases. Lets make it more clear by taking the example of protein kinase A. The serine residues of the target proteins (phosphorylase kinase, CREB) which are phosphorylated by PKA are usually dephosphorylated by phosphatase called as protein phosphatase 1. Thus, the levels of phosphorylation of the target proteins which are phosphorylated by PKA is counter-balanced by the activities of protein phosphatases (diagram of regulation of phosphorylation by PKA and protein phosphatase 1 shown below).

Just note that although most of the effects of cAMP are mediated by PKA. However, cAMP can also directly regulate ion channels with no requirement of protein phosphorylation. 

Types of Receptors - Part 4

As we have seen in the previous post that the majority of the receptors are enzyme linked that stimulate the protein-tyrosine phosphorylation. Here, in this last post regarding the types of receptors, we will discuss about the other types of receptors that are associated with some other enzymatic activities. These are: protein tyrosine phosphatases, protein serine/threonine kinases and guanylyl cyclases. Lets understand briefly about each of these classes one by one below.

Protein Tyrosine Phosphatases:

Here, the name suggests the function i.e; phosphatases which is to remove phosphate group from phosphotyrosine residues. Thus, these receptors play a role opposite to that of protein tyrosine kinases thereby counterbalancing the kinases (just to recall, the kinases, add phosphate groups to the tyrosine residues). In some cases, the protein tyrosine phosphatases play a negative role in cell signaling pathways by stopping the signals which were initiated by protein-tyrosine phosphorylation. Whereas in some cases, the protein tyrosine phosphatases play a positive role in cell signaling by their enzymatic activities.

Serine/Threonine Kinases:

The receptors for some polypeptides phosphorylates serine or threonine residues instead of tyrosine residues on their substrate molecules. An example of such receptor is Transforming Growth Factor-β (abbreviated as TGF-β) which falls under this class of receptors i.e.; serine/threonine receptors. TGF-β is a family of growth factors which is involved in the control of proliferation and differentiation of various cell types. When the ligand binds, there is an association of two different types of polypeptides (both of which are encoded by different TGF-β receptor family) and hence forms the heterodimers. Here, one of the receptor kinases phosphorylates another one. This activated TGF-β receptors then phosphorylates another group of transcription factors called Smads, which then translocates to nucleus and causes the expression of targeted genes. The entire TGF-β signaling will be explained in the further post.

Guanylyl Cyclases:

Another class of receptors is guanylyl cyclases. Guanylyl cyclases have the cytosolic domain which catalyzes the formation of cyclic GMP. As we have seen earlier, that the signaling molecule, nitric oxide, also stimulates the guanylyl cyclase. However, the target of NO is intracellular enzyme as against the transmembrane receptor.
The receptor guanylyl cyclases have extracellular domain that binds the ligand, a single transmembrane α-helix and a cytosolic domain which has the catalytic activity. Thus, when the ligand binds at the extracellular domain, cyclase activity is stimulated which results in the formation of cyclic GMP - a second messenger.

So, finally, her we finish almost all the classes of receptors. Now, from the next post, we will proceed to the pathways of intracellular signal transduction.

Types of Receptors - Part 3

In this post, I will discuss about the next class of receptor - Cytokine Receptors and Nonreceptor Protein Tyrosine Kinases.
The basic principle behind the functioning of these receptors is that they stimulate the intracellular protein-tyrosine kinases. The receptors are associated with these kinases by non-covalent bonds. This is unlike protein-tyrosine kinases (discussed here) where there is intrinsic enzymatic activity. Lets understand these receptors in detail.
First of all, what all types of receptors are included in this family? So, the answer is the receptors for most cytokines (for example, interleukin-2, erythropoietin) as well as some of the peptide hormones (like, growth hormones) are included in this superfamily of cytokine receptors. Lets understand the structure and functioning of these receptors.

Structure:

The structure is similar to that of protein tyrosine kinase receptors. So, the cytokine receptors have an N-terminal, C terminal and a transmembrane. The N-terminal is the extracellular domain that binds to the ligand while the C-terminal is cytosolic domain. The transmembrane is single and is alpha helical. Now, you might be thinking, if the structure of cytokine receptors is exactly the same as that of protein tyrosine kinase receptors (described in earlier post), then why cytokine receptors are placed in a different class. OK, I will now make it clear that there is one main difference between the two types of receptors - in the cytokine receptors, there is no catalytic activity in the C terminal (cytosolic domain) as against protein-tyrosine kinase receptors which possesses tyrosine kinase activity in C-terminal. So, next question you might be wondering is how do then, cytokine receptors function? The answer is that these receptors function in association with nonreceptor protein-tyrosine kinases, which are activated when ligand binds at the N-terminal. The description below regarding the functioning of these receptors will make your concept all the more clear.

Functioning:

Receptor Dimerization: The binding of the ligand at the N-terminal induces the receptors to dimerize.
This dimerization leads to cross-phosphorylation of the associated nonreceptor tyrosine kinases (as can be seen in the third figure in the adjacent diagram explaining the functioning).
These activated non-tyrosine kinases then phosphorylates the cytokine receptor (last figure in the adjacent diagram) and thus, provide phosphotyrosine binding sites. These binding sites then recruit the downstream signaling molecules and these molecules contain SH2 domains.

So, here, if we want to compare the two receptors (cytokine receptors and protein tyrosine kinase receptors) then, we can think of the analogy that the combination of cytokine receptors plus the nonreceptor protein-tyrosine kinases functions similar to that of protein tyrosine kinases.
One of the kinases which are associated with cytokine receptors and nonreceptor protein tyrosine kinases belong to the family of Janus Kinases (JAK) which consists of four closely related nonreceptor tyrosine kinases.
Another nonreceptor protein kinases belong to the family of Src, which consists of Src and eight closely related proteins.

Types of Receptors - Part 2

In this post, I will discuss about the next cell-surface receptor which is Protein-Tyrosine Kinases. These types of cell-surface receptors are directly linked to intracellular enzymes i.e.; this is enzyme-linked receptor. The protein tyrosine kinases is the largest family of such types of receptors.

Why are receptors called so? For that, first, you must know the function of kinases?  Kinases transfer high energy phosphate groups from one molecule to another. Now, the phosphate groups from the receptor is transferred to tyrosine residues on substrate molecule. In other words, these receptors phosphorylate on tyrosine residues on the substrate proteins; and hence the name Protein-Tyrosine kinases.I hope this is clear to you.

There are a wide variety of receptors that fall under this category, like receptors for EGF (Epidermal Growth Factor), NGF (Nerve Growth Factor), PDGF (Platelet-Derived Growth Factor), insulin and many such other growth factors. All these receptors share a common structure which is described below.

Structure: 

All the receptors under protein-tyrosine kinases have a common structural organization. All these receptors possess two terminals as N-terminal and C-terminal and a transmembrane protein. The  N-terminal is the extracellular ligand-binding domain while the C-terminal is the cytosolic domain with protein-tyrosine kinase activity. The middle portion is a single transmembrane alpha helix. Although maximum number of receptors consists of single polypeptide chain, however, some receptors do have two polypeptide chains (dimers) like insulin receptor.

Functioning:
Lets make the functioning easy to remember and recall by dividing into different steps as:

Receptor Dimerization: The first step in the signaling by these protein-tyrosine kinase receptors is the dimerization of receptors. When there is binding of ligand at the N-terminus of the receptor, then the receptors dimerize. Thus, we can say that the dimerization is induced by ligands (like growth factors). The receptors of some growth factors (like EGF) are monomers and they undergo dimerization so as to result in conformational change. These conformational changes helps in protein-protein interaction between different receptor polypeptide chains. However, there are receptors of some growth factors like NGF and PDGF which are dimers and consists of two identical polypeptide chains. Here, the growth factors directly dimerize by binding to two different receptor molecules simultaneously.
Autophosphorylation: The next step after ligand-induced dimerization is autophosphorylation. The dimerized polypeptide chains cross phosphorylate one another (as can be seen in the diagram). This leads to following events:
a. As there is phosphorylation of these tyrosine residues, it increases the protein kinase activity in the catalytic domain.
b. Secondly, phosphorylation of the tyrosine residues creates specific binding sites for additional proteins outside the catalytic domain which further transmits the intracellular signals to the downstream molecules.

These downstream signaling molecules associates with the receptor (protein-tyrosine kinase) with the help of various protein domains present within the downstream signaling molecules. These protein domains are specific to phosohotyrosine containing peptides. Hence, we can say, there is association of downstream signaling molecules with the receptor; mediated by specific protein domains.

An example of one such domain which was one of the first to be characterized is SH2 domain. Why the name SH2? It stands for Src Homology 2 (where Src is an oncogenic protein) as it was initially recognized in protein-tyrosine kinases related to Src.  These SH2 domains bind to specific sequences which contains phosphotyrosine resides.
Another example of such domain is PTB domain where PTB stands for Phospho-Tyrosine Binding. There are some other proteins (which do not bind via SH2 domains) that bind via PTB domains.
The effects of protein binding to activated protein-tyrosine kinase receptor via SH2 domain or PTB domain are as follows:
a. The protein gets localized to plasma membrane
b. There is association of several other proteins with protein/s
c. Promotes the phosphorylation of several other proteins
d. Ultimately stimulates their enzymatic activity

Types of Receptors - Part 1

Before understanding the pathways and mechanisms of complicated cell-cell signaling, lets make it easy by first understanding the different types of cell receptors and how do they function. Understanding this, will make the cell-cell signaling very clear and easy to remember.

From our previous posts, we can recall that cell-cell signaling requires binding of the signaling molecules to the cell-surface receptors on the target cells. So, lets first understand different classes of cell surface receptors with their functioning. In the next few articles, we will be learning about classes of cell surface receptor then followed by the pathways and the mechanisms of signaling and communication (downstream signaling).

Lets start our discussion with the first family of cell receptors which is also the largest - G-Protein Coupled Receptors, abbreviated as GPCRs. You might be thinking what is this "G-protein"? The G-protein stands for guanine nucleotide binding protein. The G-protein coupled receptors or GPCRs utilizes these G-proteins as an intermediate to transmit signals to intracellular targets. There are around thousands of GPCRs. Do you know, that these receptors are also responsible for our various senses like smell, sight and taste? Isn't it interesting to know how it works? Before that, lets understand the structure of GPCRs.

Structure:
The G-protein coupled receptors consists of proteins which are characterized by seven α-helices (as can be seen in the adjacent figure) that span the membrane and hence are called membrane-spanning α-helices.

G proteins consists of three subunits as α, β and γ. They are also referred to as heterotrimeric G proteins. What is the role of these subunits, we will see in the functioning of the receptor. Before understanding the functioning, just remember:
α-subunit bound to GDP ---- Inactive State

α-subunit bound to GTP ---- Active State

Functioning: 
The α-subunit binds to guanine nucleotide which regulates G-protein activity. In the resting stage, the α-subunit is bound to GDP in association with β and γ subunits (here, α-subunit bound to GDP is inactive state) (in the adjacent figure - the topmost diagram). When the hormone binds to the extracellular domain of these receptors, there is a change in the conformation of the receptors such that the cytosolic domain of the receptor interacts with the G-protein. This leads to the release of GDP from α-subunit and in turn it gets exchanged with GTP. This α-subunit which is now bound to GTP (active state), dissociates with β and γ subunits and these two subunits remain together as βγ complex (the left diagram in the above figure).
The α-bound GTP and βγ complex functions by interacting with their respective targets and gives an intracellular response. When there is hydrolysis of GTP, the activity of  α-subunit is terminated and this inactive α-subunit (which is bound to GDP) then re-associates with βγ complex and the cycle starts again.

There is a wide range of α, β and γ subunits. For example, mammalian genome codes for around 20α subunits, 5β subunits and 12γ subunits. Different G proteins associate with different receptors and hence, there are distinct intracellular targets.

(Note: What makes it binds to GTP after conformational change. GDP (guanine dinucleotide phosphate has two phosphate groups while, guanine trinucleotide phosphate, GTP has three phosphate groups. So, when there is an active state (conformational change), the conformation is such that it can accommodate 3 phosphate groups and hence GDP is replaced by GTP.

All About Signaling Molecules

As we have seen in the previous post regarding some of the signaling molecules that transmit information. In this post, I will clear out the different types of signaling molecules. Lets classify the different signaling molecules in five different categories as below:

1. Steroid Hormones and Nuclear Receptor Superfamily:

As we have seen in the previous post that the signaling molecules bind to receptors which are present on the surface of the target cell. However, there are certain molecules that are intracellular and are present in the cytosol and the nucleus. It is interesting to know that these intracellular molecules respond to various small hydrophobic signaling molecules which diffuse the cell membrane. Let me make this more clear to you by taking the examples of steroid hormones.  All the steroids hormones including testosterone, estrogen, progesterone, corticosteroids and ecdysone are included under this sub-heading of steroid signaling molecules. The former three are sex steroids which are produced by gonads and are synthesized from cholesterol (structures can be seen in the adjacent figure). The corticosteroids are produced by adrenal gland. These include glucocorticoids and mineralocorticoids (representative structure in the above figure). The glucocorticoids act on variety of cells to stimulate the production of glucose whereas the mineralocorticoids act  on kidney to regulate water and salt balance. Ecdysone is an insect hormone and it plays a key role in the metamorphosis from larvae to insect. The brassinosteroids are plant-specific steroids that help in a number of processes like plant differentiation, cell growth etc.

These steroid hormones are hydrophobic in nature and hence they can cross the plasma membrane by diffusion. When they enter inside the cell, they bind to intracellular receptors. Now, what are these intracellular receptors? These receptors are nothing but the receptors which are expressed by the target cells which are responsive to the hormones. These receptors are the members of nuclear receptor superfamily and are transcription factors which includes domains for ligand binding. This ligand binding can act as activators or repressors thereby regulating the function. Hence, the steroid hormones directly regulates the gene expression.
Lets take an example of estrogen receptor and understand how the ligand binding has distinct effect. When the hormone estrogen is not present, the estrogen receptor is bound to chaperone, Hsp90 (as can be seen in the adjacent figure). In the presence of estrogen, there is conformational change, as a result of which Hsp90 is displaced and it leads to the formation of receptor dimers which then further binds to regulatory DNA sequence and hence regulates gene expression.

Just similar to these steroid hormone functions the thyroid hormone, vitamin D3 and retinoic acid (structure of these molecules in the adjacent figure).

2. Nitric Oxide and Carbon Monoxide:

Nitric oxide (NO) and Carbon Monoxide (CO) both are simple gases and functions as signaling molecules. NO is a paracrine signaling molecule in nervous, immune and circulatory systems. Being a small molecule it can easily diffuse the plasma membrane. However, a point to note is that it doesn't behave like steroid hormones (recall that, steroid hormones bind to a receptor that regulates transcription), NO rather alters the activity of intracellular target enzymes. Points to remember:

i. NO is synthesized inside the cell from the ariginine. Once produced, it then diffuses out of the cell and can act locally on nearby cells. This is so because NO is extremely unstable as half life of NO is only for a few seconds.

ii. The major intracellular target of NO is guanylyl cyclase. NO binds at the enzyme's heme group and stimulates the synthesis of cyclic GMP.

iii. NO directly modifies some target proteins by nitrosylation of cysteine residues. 

iv. NO also signals the dilation of blood vessels.

CO functions as signaling molecule in nervous system. It is closely related to NO and acts similar to that of a neurotransmitter and helps is blood vessel dilation.

3. Neurotransmitters:

Neurotransmitters are a group of small molecules that are hydrophilic in nature (hence unable to cross the plasma membrane of the target cell). These include acetylcholine, dopamine, adrenaline, serotonin, histamine, glutamate, glycine and GABA (structures of all these are given in the adjacent figure).

The neurotransmitters, as the name suggests, transmits or carries the signals between neurons or from one neuron to other parts of the cell. When there is action potential at the terminus of the neuron, the neurotransmitters are released. They diffuse through the synaptic cleft and binds to the receptor on the target cell.
Note: Unlike steroid hormones, NO and CO, the neurotransmitters act by binding to the receptor at the cell surface.

4. Peptide Hormones and Growth Factors:

The maximum number of signaling molecules fall under this group of peptides which ranges from a few amino acids to more than a hundred amino acids. The peptide hormones includes neuropeptides and a variety of growth factors. Neuropeptides are the small peptides which are released by neurons instead of  neurotransmitters. The peptides include enkephalins and endorphins which acts as neurotransmitters as well as neurohormones i.e.; not only they function at synapses but also at distant cells. The enkephalins and endorphins act as natural analgesics that decreases pain.
The various polypeptide growth factors include:

i. Nerve Growth Factor (NGF) - Regulate the survival and regulation of neurons.

ii. Epidermal Growth Factor (EGF) - Stimulates cell proliferation

iii. Platelet derived growth factor (PDGF) - Helps in wound healing.

iv. Cytokines - Regulates the development of blood cells and control activity of lymphocytes

v. Membrane-anchored growth factors - Remains associated with plasma membrane.

All these hormones and growth factors are unable to cross the plasma membrane and hence they act by binding to the receptor at the cell surface. 

5. Eicosanoids:

Eicosanoids is a class of lipids that serves a signaling molecules. This group includes prostaglandins, prostacyclins, thrombaxanes and leukotrienes. They affect the target cells in various ways like blood platelet aggregation, inflammation and smooth muscle contraction. These eicosanoids are broken down rapidly and they act locally in paracrine or autocrine signaling.

Cell-Cell Signaling

Have you ever wondered, how do cells talk with each other or how do they communicate? How do they come to know what has to be done if you get hurt or if you are hungry or thirsty? Isn't it very interesting to know that all types of cell receive information and respond to signals from their environment. But you might be wondering, how? The answer is through ‘Signaling Molecules’ which play a lead role in this process of cell-cell communication. Now, you might be thinking, what are these signaling molecules? Where do they come from? How do they work? Ok..lets understand all about signaling molecules. These signaling molecules are basically chemicals (like nitric oxide etc.) or proteins (hormones etc.) which are secreted or expressed on the surface of the cell. They then bind to receptors which are present on the other cell (these cells are called target cell)  or sometimes even present on the same cell; thereby coordinating the functions of various cells. The binding of these signaling molecules on the receptor creates a series of reactions that regulates various methods/systems like movement, metabolism, survival, differentiation etc. The breakdown of these pathways has resulted in various types of cancer and hence has become a very interesting field to study.

Modes of Cell-Cell Signaling:

Signaling by Direct Cell-to-Cell Interaction

Cell signaling can take place by direct cell-to-cell interaction (as can be seen in the adjacent figure) where, on the surface of one cell, is the signaling molecule that binds directly to receptor, which is present on the surface of the other cell. The cell signaling/ cell-cell communication can also take place by the action of various signaling molecules. As already mentioned above, various cell receptors are present on the target cells which bind to signaling molecules.
The different types/modes of signaling by secreted molecules are mainly of three types:

Diagrammatic Representation of Endocrine Signaling

1. Endocrine Signaling: Is it difficult to remember these different types of signaling with their functions? Don't worry! I will try to make it easy for you - split the word and understand the  meaning of individual word, then its easy to remember these words with their functions. So, lets understand the meaning of endocrine. The word “endocrine” originates from two words, 'endo' and 'crine'. The former word means 'within' and the suffix 'crine' means 'to secrete' or 'separate' Thus, the word endocrine means ‘secrete from within’. In this type of signaling, the signaling molecules are secreted from within the endocrine glands; they travel through the circulation system and act on/ bind to receptor molecules on the target cell (as can be seen in the above figure). The signaling molecules in this type of signaling are ‘hormones'. Hormones are secreted by specialized endocrine cells of endocrine glands which act on target cells which are at distant and reach via the circulation system. An example of endocrine signaling: The hormone, insulin, is secreted by pancreas which then travel through the circulatory system which has an effect of stimulating muscle cell or adipose cells.

Diagrammatic Representation of Paracrine Signaling

2. Paracrine Signaling: 'Para' means 'nearby' and 'crine' means ' to secrete'. Thus, 'paracrine' word means 'secretion nearby'. Thus, in this type of signaling, the molecules (signaling molecule) released by one cell acts on or bind to the receptor on the nearby neighbouring cell. A very common example of paracrine signaling is the neurotrasmitters. These are present at the synapse  or junction of neuron cells and help in transmitting the signals. So, neurotransmitters are released by one neuron cell and they act on nearby neuron cell to transmit the signal.

Diagrammatic Representation of Autocrine Signaling

3. Autocrine Signaling: 'Auto' means 'self' and 'crine' means 'to secrete'. Thus, 'autocrine' means 'secrete to itself' Thus, in this type of signaling, the molecules (receptor molecules) are secreted from the cell and act on the same cell. In other words, some cells respond to signaling molecules that they themselves produce.
For example, the cytokine, Interleukin-1 (IL-1) in monocytes. The interelukin is produced in monocytes response to some external stimuli and it binds to receptor present on the surface of its own cell. 


I hope now you will remember these different types of signaling.