Signal Transduction: Getting the Message Across

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The membrane as an information barrier

So far, we have dealt with the plasma membrane as a physical barrier that allows the cell, through the processes of homeostasis, to maintain a comfortable internal environment. All well and good, but cells must also be able to adapt to changes in their external environment and the cells of a multicellular organism must be able to alter their function in a co-ordinated way in order to serve the needs of the entire organism. The key word here is co-ordinated. Imagine what would happen if all the cells of the renal collecting duct changed their water permeability independently rather than under the control of ADH. Skeletal muscle fibres are individually capable of very little, but the co-ordinated action of all the fibres is what enables us to walk and talk and and write extensive learning support notes.

Co-ordination is achieved by cells and organs communicating with one another. If you want to raise your right arm, your brain must communicate your intent to the muscles of your right arm. The signal will be initiated by electrical activity and the release of signalling molecules in the brain, the signal will be actively tramsitted through nerves and your muscles will be activated by a release of signalling molecules. The final extracellular step in just about any signalling pathways (neural, endocrine, apocrine etc.) is the release of a signalling molecule at the surface of the target cell. Most signalling molecules are small and water soluble (the notable exception are steroid hormones), exactly the sort of thing that the membrane is supposed to keep out of the cell. The cell must, therefore, allow the signal to cross the membrane into the cell whilst continuing to exclude the signalling molecule. This is what receptors are for. A receptor is a cunningly shaped protein molecule embedded in the plasma membrane that binds only a single type of signalling molecule and is able, one way or another, to let the inside of the cell know that something has bound to it.

First messengers

The signalling molecule released at the surface of the target cell is called a First messenger, presumably because it is the first step in the signal transduction process. There are many, many first messengers. You should already know about acetylcholine (ACh) and noradrenaline (NA, norepinephrine) which are respectively the primary neurotransmitters in the parasympathetic and sympathetic branches of the autonomic nervous system.

Receptors

There must be at least one type of receptor for each first messenger substance (otherwise it isn't a signalling molecule). Many first messengers have more than one type of receptor, so there are (many) more receptor types than there are first messengers. For example, there are two main classes (and several subtypes) of the ACh receptor; nicotinic and muscarinic. Other than the fact that both receptors bind ACh, they have almost nothing else in common! The major types and subtypes of adrenergic receptors are α1, α2, β1, β2 and β3

Second Messengers

The most common way of passing the 'message' into the cell is for the activated receptor to generate an intracellular signalling molecule. In an astonishing display of consistency, these intracellular signalling molecules are called second messengers. There are probably not as many 2nd messengers as there are first messengers, but there are still a lot. Calcium and cyclic adenosine monophosphate (cAMP) are the best known and the most studied. The function of the 2nd messenger is bring about a change in the biological activity of the cell.

Signal Transduction

Together, these three processes;signal transduction

  1. Release of a 1st messenger
  2. Binding of the first messenger to a receptor and generation of a 2nd messenger
  3. A change in the biological activity of the cell brought about by the 2nd messenger

comprise signal transduction.

One of the fundamentals of signal transduction is that the change in biological activity depends on the type of a cell. This might seem so obvious that there is no need to say it; there is no way, for example, that anyone could imagine that a neurone and a hair cell responding in the same way to stimulation. BUT confusion is possible because neurone and hair cell have at least one receptor type in common and they both have the same 2nd messenger pathways. No matter whether they use the same receptor, or the same 1st messenger or the same second messenger, the end result, the change in biological activity will be different.

Don't panic.... BUT

There are lots of 1st messengers; for example

are all 1st messengers (and there are lots more).

There are lots and lots of receptors. The only reason that receptor biochemists are able to preserve any vestige of sanity is because receptors belong to families and superfamilies. This is important because, if you know how one member of a family behaves, then you can make some pretty good guessess about the behaviour of the rest of the family. The muscarinic ACh receptor belongs to the family of G-protein coupled receptors which also includes receptors that bind noradrenalin, calcitonin, dopamine, histamine, prostaglandins and anti diuretic hormone (to name but a few). These are all different receptors that bind different 1st messengers and trigger a range of 2nd messengers BUT the way that they trigger production of the second messenger is the same in every case. The nicotinic ACh receptor belongs to the the ion-channel linked (ionotropic) superfamily of receptors. Two other receptor superfamilies are based on the common ability to activate either an intrinsic (e.g. the insulin receptor) or a soluble (e.g. cytokine receptors) tyrosine kinase.

There aren't as many 2nd mesengers as there are first messengers, but.... a list of common 2nd messengers would include

Really, don't panic

I have listed all these first messengers, receptors and second messengers so that you know that there are lots and so that you don't get the idea that ACh, NA, cAMP, IP3 & Ca2+ are all that there is. A detailed knowledge of the components and workings of every signal transduction pathway is beyond the needs and requirements of the average medical or dental student. It is, however appropriate that you know that they exist. I will concentrate on ACh, NA, cAMP, IP3 & Ca2+ and if you can understand what these things do and how they do it then you will have no difficulty picking up on other signalling processes as and when (and if) you need to.

Examples

I think that the best way of explaining intracellular signalling in more detail is to use some examples. Three in fact. The first is excitation contraction coupling in skeletal muscle, the second is protein secretion in salivary gland cells and the third is fluid secretion in salivary gland cells. There are reasons for choosing these examples. First, muscle cells are capable of action potentials and salivary gland cells aren't. From an intracellular signalling point-of-view, this is a pretty fundamental difference. The mechanics of the signalling process in excitable cells can be very different from that in non-excitable cells. I also have several reasons for choosing salivary gland cells as my example of a non-excitable cell. One reason is that I research signalling in salivary gland acinar cells, so I know a little bit about it. Another is that dental students must know about salivary glands, so this represents an opportunity for some early exposure. Most important, salivary gland cells have two second messenger pathways, one controlling protein secretion and the other fluid secretion so we can look at two, almost independent, pathways in the same cell.

Excitation-contraction coupling in skletal muscle

The contractile machinery in muscle cells is Ca2+ dependent. If cytoplasmic Ca2+ concentration increases then the muscle contracts. All we need to know, is what causes intracellular Ca2+ to increase.

Here is the sequence of events

    excitration contraction coupling
  1. The nerve controlling the muscle releases acetylcholine (1st messenger) at the neuromuscular junction
  2. Acetylcholine binds to a nicotinic acetylcholine receptor, which is also a Na+ channel, causing it to open.
  3. Influx of Na+ through the nicotinic acetylcholine receptor/channel depolarises the membrane from a resting value of -70mV.
  4. If the membrane potential falls below -55mV then an action potential is triggered and the membrane depolarises explosively, reaching about +20mV
  5. The depolarisation activates voltage sensors on Ca2+ channels on the 't' tubules of the plasma membrane
  6. As a result of activation of the voltage sensors, The Ca2+ channels on the 't' tubles AND Ca2+ channels on the sarcoplasmic reticulum open
  7. Intracellular Ca2+ activity increases because the concentration of Ca2+ inside the sarcoplasmic reticulum and outside the cell is much higher than the concentration of the cytoplasm, so Ca2+ moves down its concentation gradient.
  8. Contraction follows

Ok, so what is the point? Why are there so many steps simply to make a muscle contract? I think that the simplest (best?) explanation is that the cell needs an action potential for synchronisation. An action potential conducts instantaneously through the whole cell. Once the action potential is triggered then the whole of the muscle cell will contract. If you accept the need for the action potential, then the point of the other processes become obvious. The nicotinic ACh receptor triggers the action potential and the voltage gated Ca2+ channels respond to the action potential.

Signal transduction in salivary glands

salivary secretionSalivary glands produce saliva, a watery secretion containing protein including the digestive enzyme salivary amylase. Secretion occurs in two parts. Proteins are secreted by exocytosis which is under the control of the sympathetic nervous system and transduced by noradrenalin and cyclic AMP. Fluid (and electrolyte) secretion is controlled by the parasympathetic nerves using acetylcholine and Ca2+ as first and second messengers respectively. This is a fine example of the sympathetic and parasympathetic branches of the nervous system working in cooperation, not in opposition.

Stimulus/secretion coupling in salivary acinar cells

I don't want to get bogged down in the mechanism of fluid secretion, suffice it to say that the process is Ca2+ dependent and the key target of the increase in Ca2+ is activation of an apical membrane Cl- channel. Anyway, if the the Ca2+ concentration in the cell increases, fluid secretion will occur. Here is the sequence of events that lead to an increase in the Ca2+ concentration in the cytoplasm of salivary gland acinar cells.

    7 membrane domain G protein linked receptor
  1. Parasympathetic nerves release ACh close to the acinar cells
  2. ACh binds to a muscarinic (seven-membrane-spanning-domain-G-protein-activating) receptor.
  3. GTP replaces GDP on the α-subunit of a heterotrimeric G-protein associated with the cytoplasmic domain of the membrane. (This is the step that passes the signal across the plasma membrane even though nothing physical at all has crossed.
  4. The activated α-subunit of the G-protein disassociates from the β and γ subunits and activates a membrane bound enzyme, phospholipase C (PLC)

    PLC etc
  1. Phospholipase C hydolyses phosphoinositol 4,5 bis phosphate (PIP2) into diaclglycerol and inositol 1,4,5 trisphosphate (IP3). (Diacylglycerol remains within the plasma membrane and is a second messenger in its own right causing activation of protein kinase C.)
  2. IP3 leaves the plasma membrane and moves into the cytoplasm and binds to IP3 receptors on the endoplasmic reticulum. These receptors are also Ca2+ channels and IP3 binding opens them.
  3. The activity of Ca2+ inside the endoplasmic reticulum is much higher than that of the cytoplasm, so Ca2+ moves down its concentation gradient and intracellular Ca2+ activity increases.

This very simplified schematic leaves several things unresolved.

What is the point of all this? There are many possible points. One is synchronisation, or rather the lack of it. If you stimulate these cells with a high concentration of ACh then you get an elevation of Ca2+ across the whole cell, however this Ca2+ signal moves across the cell in a stately progression from the apical to the basolateral pole as a wave, taking about 100ms to get from one end to the other. If you stimulate the cells gently, then you get a high frequency oscillatory Ca2+ signal in the apical pole only. The apical pole is the "business" end of the cell, this is where secretion is happening, It makes sense to have a signal that stays there. Another, very, very important point is amplification. There are two potential amplification points in this system. The most important is at the phospholipase C (PLC) stage. PLC is activated by the active α subunit of the G-protein. The "off-switch", hydrolysis of GTP by the α subunit, is slow and so PLC has time to manufacture many IP3 molecules every time it is activated. Similarly, for as long as ACh is bound to the ACh receptor, the β γ subunits of the G-protein are active and they could activate an another α subunit, should one bind to them.

Protein secretion in salivary acinar cells

Here we go again. Protein secretion follows an increase in cyclic adenosine monophosphate (cAMP) concentration. The steps involved in increasing cAMP concentration are as follows.

    7 membrane domain G protein linked receptor
  1. Sympathetic nerves release noradrenalin (NA) close to the acinar cells
  2. NA binds to a β-adrenergic (seven-membrane-spanning-domain-G-protein-activating) receptor.
  3. GTP replaces GDP on the α-subunit of a heterotrimeric G-protein associated with the cytoplasmic domain of the membrane.
  4. The activated α-subunit of the G-protein disassociates from the β and γ subunits and activates a membrane bound enzyme, adenylate (adenyl) cyclase




    adenylate cyclase and cAMP
  1. Adenylate cyclase converts ATP into cAMP

This simplified schematic also leaves things unresolved.

This schematic also reveals another "point" to signal transduction mechanisms. The first 4 steps of signal transduction in both fluid and protein secretion are identical. The receptors belong to the same family (seven-membrane-spanning-domain-G-protein-activating) and, as promised, behave in the same way. Ok, so the domain that binds the neurotransmitter and the associated G-proteins are different. The signal amplification identified in stimulus-secretion coupling works just as well in this signalling pathway.

And finally... almost, what step of the protein secretion process does cAMP switch on? Answer, all steps. cAMP stimulates gene transcription, post-transcriptional modification, maturation of secretory vesicles and exocytosis. This sort of global switch-on of many processes directed towards a single goal (in this case, protein secretion) is characteristic of cAMP-mediated second messenger pathways.

Conclusions

"Second messengers couple external stimulus to change in biological activity". This simple, accurate statement conceals the incredible sophistication of signalling processes. Each type of biological activity, for example, muscle contration, fluid secretion or protein secretion is regulated by the signaling pathway to which it is intrinsically best suited. An action potential-mediated process is the perfect regulator for muscle contraction which is an all-or-none sort of process. The cell contracts or it doesn't. You don't want only one end of the cell to contract, you don't want any lag between contraction at one end of the cell and contraction at the other. Fluid secretion is a different sort of process in which there may be benefit in differentially regulating the apical and basolateral ends of the cell. IP3 driven Ca2+ signalling allows for local increases in Ca2+ that don't spread across the cell. Control of protein secretion requires many processes to be switched on, all at once. Cyclic AMP is particularly good at this.

These notes are an INTRODUCTION to signal transduction and 2nd messengers that barely scrapes the surface of a subject that has been (and still remains) a "hot" research area for over 15 years. To the best of my knowledge, everything written here is true and pretty much up to date. However, I have left out many many things. There is a more detailed "take", for anyone who is interested, on fluid and protein secretion in the salivary secretion notes.