Signalling molecules (memorising )

Types of Signalling Molecules

Signalling molecules can be classified based on their biophysical properties. Molecules are typically either hydrophilic (meaning soluble in aqueous solutions such as blood and lymph), or hydrophobic (meaning insoluble in hydrophilic solutions). Hydrophilic signalling molecules are able to travel directly in the blood, extracellular fluid or lymph however hydrophobic signals require solubilisation by transport proteins in order for them to be secreted and travel to their target cell.

Generally speaking, hydrophobic signals function by targeting intracellular (nuclear) receptors, whereas hydrophilic receptors require a plasma membrane bound receptor, as the signal molecule is unable to pass through the lipid bilayer.

Classification of Signalling Molecules

There are six major classifications

• Peptides – Hydrophilic

• Amines – either Hydrophilic or Hydrophobic*

• Steroids – Hydrophobic

• Lipids – Hydrophobic

• Purines – Hydrophilic

• Gases – Hydrophilic

*each molecule is either hydrophobic or hydrophilic, but the class contains both types of molecules

The first three classes make up all known hormones and most of the known neurotransmitters.

Peptides

For the sake of classification, peptides will include the single amino acids which can function as neurotransmitters. These are Aspartic Acid, Cysteine and Glutamic Acid. Glutamic acid is processed via decarboxylation to form GABA.

The true peptides (i.e. those which contain at least one peptide bond) require at least two amino acids. Examples of these true peptide signals include the neurotransmitter endorphin and the hydrophilic hormones such as oxytocin.

All of the peptide signal molecules are translated from mRNA in the rough Endoplasmic Reticulum and are subject to post translational modification in the Golgi Apparatus. Examples of such post translational modifications include glycosylation, where carbohydrate groups are added to a protein following its translation. These proteins are packaged into secretory vesicles so they can be released into the extracellular space via the process of exocytosis. Release may either be immediately after synthesis, such as in the case of cytokines, or the signal may be stored for long periods of time, only being released when required. Examples of signals which are stored for use this way include peptide hormones and neurotransmitters.

Release of these signalling molecules occurs via exocytosis. The secretory vesicle fuses with the cell’s plasma membrane, and in doing so the contents of the vesicle end up on the outside of the cell. These peptide signals are all soluble in water and do not require any transportation or carrier protein in order to circulate in an aqueous solution. 

Peptide signals act via cell surface receptors and may have a number of different effects. If the membrane receptor is an ionotropic receptor, the binding of the signal to the receptor results in a change in ion flux into the cell. By contrast, if the receptor is a metabotropic receptor, the binding of the ligand to the receptor results in a change in an enzyme’s activity within the cell.

In order to stop the signal from activating the receptor continually, protease/peptidase enzymes are present to degrade the signal. For simple peptide signals, this degradation is rapid and so the signal may have a half life of only a few seconds. In contrast, glycoproteins are more complex and are degraded more slowly, yielding a half life measured in hours. As a consequence of this signal turnover, sustained signal effect requires that the signal must be continually synthesised by the signalling cell.

Biogenic Amines

These molecules are those which contain an amine group (NH₂) and are usually derived from an amino acid. The following table summarises the biogenic amines

The Catecholamines are derivatives of Tyrosine and are all hydrophilic. Examples of these types of signalling molecules include Dopamine, a neurotransmitter found in all taxa, Octopamine, a neurotransmitter found in invertebrates), Noradrenaline (a vertebrate neurotransmitter) and adrenaline (both a paracrine and endocrine hormone found in vertebrates.

Indoleamines are derivatives of tryptophan and are hydrophilic. Examples of these types of biogenic amine include Serotonin (also known as 5HT) which act via 5HT receptors on the plasma membrane. 5HT can also be metabolised into melatonin which is a neurotransmitter in most taxa and associated with the regulation of circadian and seasonal rhythms in vertebrates.  It has a strong association with Seasonal Affective Disorder (SAD).

Histamines are amine metabolites of histidine. Histamine is a neurotransmitter and also a paracrine signalling molecule in all taxa. It acts via a number of different plasma membrane receptors, each of which elicits different responses and are found in different tissues. In the vascular tissue, histamine results in vasodilation whereas in the bronchus, histamine results in bronchoconstriction. Is also a well known chemoattractant for mast cells and is strongly associated with allergic responses.

Acetylcholine is a well known neurotransmitter and metabolite of choline . It is found in all taxa and acts via the plasma membrane receptors. Nicotinic ACh receptors are ionotropic and respond to signal binding by allowing ion flux into the receiver cell. Metabotropic ACh receptors function by altering the metabotropic receptor GPCR.

The thyroid hormones are unique to vertebrates and are all hydrophobic. Only a very small proportion (<1% is free within the blood) as the majority requires a carrier protein in order to be soluble within an aqueous fluid. Examples of such carrier proteins include Thyroxine Binding Globulin and Albumin, amongst others. They function by activating the a nuclear receptor which increases basal metabolic rate.

Steroids

These signal molecules are all derivatives of cholesterol, a hydrophobic 27 carbon molecule.

They are an important class of endocrine and paracrine hormones in all vertebrates (sex hormones) and many invertebrates (ecdysone). They are also implicated as being used as pheromones for communication between organisms.

There are five classes of steroid hormone.

These molecules are all synthesised in the mitochondria and the smooth endoplasmic reticulum. They are all lipophilic, which means they are soluble in the plasma membrane and therefore cannot be stored within vesicles or within a cell. For this reason, they must be synthesised immediately prior to their secretion from a cell.

As these molecules are all hydrophobic, they are insoluble in the aqueous fluids which surround cells. In order to travel to their site of action therefore, they require binding to a carrier protein which are commonly binding globulins or albumin

The binding of the steroid hormone to the carrier protein is regulated by the laws of mass action and mass action equilibriums. This states that:

M+C⇔MCM+C⇔MC

Where M is the messenger molecule and C is the carrier molecule and MC is the messenger/carrier complex

Following these laws it can be understood that at the source of a signal, where the signal concentration is highest, most of the chemical messenger will bind the carrier protein. These proteins are contained in the circulatory system and so will move away from the source quickly to areas where the unbound messenger concentration is much lower. This results in unbinding of the messenger from the messenger-carrier complex (MC). In the unbound state again, the messenger can interact with the target cell.

Classically these steroid signals bind nuclear receptors which may be found in the cytosol (class I nuclear receptors) or within the nucleus (class II nuclear receptors) and in complex with the receptor, they function as a transcription factor. These are known as ligand dependent transcription factors and are able to bind DNA and regulate gene expression via interactions with the RNA polymerase

Thus far, you have examined the major signalling molecules but there are also rarer molecules. This short, text introduces the three less common families of signalling molecules:

• lipids;

• purines;

• gases.

It’s important to appreciate that just because the three classes of signalling molecule listed above participate in a lower proportion of cell signalling events, being less prevalent doesn’t make the lipids, purine and gaseous signalling molecules less important.  

LIPIDS

By definition, lipids are hydrophobic (water insoluble) signalling molecules, and of these, the most important would seem to BE the eicosanoids: 20 carbon (C20) lipids which include the:

• prostaglandins (PG’s) and thromboxanes (TX’s) – synthesised by the action of cyclooxygenase (COX) / prostaglandin synthase (PTGS) enzymes;

• leukotrienes (LT’s), hydroxyeicosatetraenonic acids (HETE’s), hydroperoxyeicosatetraenonic acids (HPETE’s), and lipoxins – synthesised by the lipoxygenase (LOX) enzymes.

Of the above, the prostaglandins (so called because they were originally identified as being produced in the prostate gland) and the leukotrienes (molecules made in leukocytes characterised by three C=C double bonds – hence “…trienes”) are important mediators in the inflammatory cascade where they signal to immune cells and the vasculature.

The lipid substrate for the cyclooxygenase and lipoxygenase pathways is the polyunsaturated fatty acid, arachidonic acid (AA).  Because this 20C fatty acid has 4 double bonds, all cis, it is represented by the notation C20:4. 

AA is stored esterified (via its carboxylic acid group) to glycerol in membrane phospholipids, and so the first step is the synthesis of all eicosanoids is to hydrolyse that ester bond, and hence liberate that AA substrate from membrane phospholipids, by the action of phospholipase A₂ (PLA₂).

As noted above, the eicosanoids (particularly prostaglandins and leukotrienes) are important cell signals in the inflammatory cascade, mediating both inflammation and nociception (pain signalling).  Hence, to limit inflammation and / or relieve pain, it is possible to either:

• inhibit PLA₂ using anti-inflammatory steroids – such as the glucocorticoid steroid hormone cortisol, or synthetic glucocorticoids (e.g. hydrocortisone / dexamethasone / betamethasone / methylprednisolone), or

• inhibit the downstream PTGS / COX enzymes using non-steroidal anti-inflammatory drugs (NSAID’s), such as aspirin, ibuprofen or paracetamol.

Each of the prostaglandins synthesised from AA via the common intermediate, PGH₂, has a very specific effect.  For example, while PGF_2α induces the contraction of smooth muscle (e.g. in the vasculature and uterine myometrium), PGE₂ (which differs only in having a ketone rather than a hydroxyl group at carbon position 9), relaxes smooth muscle, and so acts as a vasodilator (rather than a vasoconstrictor).

Although lipids, prostaglandins bind to and act via cell surface receptors that are G-protein-coupled receptors (GCPR’s):

• PGF_2α preferentially binds to and activates PTGFR (FP) receptors which act via the G_q G-protein to stimulate phospholipase C (PLC) culminating in the generation of the intracellular second messenger inositol-1,4,5-trisphosphate (IP₃) which, in turn, elevates the intracellular calcium (Ca²⁺) concentration to induce muscle contraction;

• PGE₂ preferentially binds to and activates PTGER (EP) receptors, of which there are 4 cloned isoforms (PTGER1, PTGER2, PTGER3 and PTGER4).  Of these, PTGER2 and PTGER4 act via the G_S G-protein to stimulate adenylyl cyclase (AC) culminating in the generation of the intracellular second messenger cyclic adenosine-3’,5’-monophosphate (cAMP) which activates protein kinase A (PKA) to mediate the intracellular response to PGE₂.   

The biological functions of all prostaglandins are reliant on the presence of a hydroxyl (alcohol) group at carbon position 15, and so PG’s are rapidly inactivated by the short-chain alcohol dehydrogenase (SCAD) enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH).  This rapid enzymatic inactivation of PG’s (caused by converting the pivotal OH group at carbon 15 to a double-bonded ketone) severely limits the range of prostaglandin action, such that they can only act as autocrine / paracrine signalling molecules, rather than as endocrine hormones – they would be rapidly inactivated if secreted into the circulation by the PGDH enzyme.

PURINES

The two purines, adenine & guanine, can exist either as nucleosides (adenosine & guanosine) or as nucleotides (e.g. AMP, ATP & GTP).  In either form, they are known to act as neurotransmitters and as neuromodulators (i.e. they modulate the neuronal response to other neurotransmitters) as well as acting locally as autocrine / paracrine hormones.  For example, in mammals, both adenosine and extracellular ATP have been show to exert a wide range of physiological actions including:

• Acting as an excitatory neurotransmitter to depolarise neurones;

• Lowering heart rate and hence cardiac output by signalling to cardiomyocytes;

• Autocrine action in leukocytes to modulate their activation as part of the immune response

• Paracrine actions on osteoblasts and osteoclasts to remodel bone (only in vertebrate taxa).

As with prostaglandins, the range of action of purines is limited by their metabolism.  The purine nucleotides are all subject to hydrolysis by ectonucleotidase enzymes (which remove sequential phosphates from the nucleotide tri-, di- and monophosphates).  It is this susceptibility to metabolism that limits the purines to exerting autocrine and paracrine (rather than endocrine) actions, or acting as neurotransmitters across the synaptic cleft for purinergic neurones.

In terms of their cellular mechanisms of action, purines activate transmembrane signalling by binding to (and hence inducing conformational changes in) purinergic receptors.  Adenosine and ATP have been shown to act both via metabotropic GPCR’s (specifically the P1 and P2Y receptors) and also by ionotropic receptors / ligand-gated ion channels: the P2X receptors.  The latter, which mediate the transmembrane flux of sodium (Na⁺), potassium (K⁺) and calcium (Ca²⁺) cations on binding extracellular ATP, have been implicated in a broad range of physiological processes, from mediating nociception (particularly in chronic pain syndromes) and platelet aggregation through to contraction of the vas deferens during ejaculation in mammals.

GASES

Gas molecules that play roles in cell-cell communication include carbon monoxide (CO) and hydrogen sulphide (HS), but the best understood example is nitric oxide (NO).  Although gas molecules are hydrophilic, they are also very small such that they can diffuse freely through the hydrophobic core of the phospholipid bilayer of the plasma membrane.  As a consequence, despite being hydrophilic, gas molecules signal by interacting with intracellular proteins (rather than cell surface receptors).

NO is synthesised from the amino acid substrate arginine (L-Arg) by NO synthase (NOS) enzymes.  NO has a very short half-life of between only 2 and 30 seconds, and so can only act locally in an autocrine / paracrine manner: it doesn’t remain active long enough to travel any distance and exert an endocrine action.

While NO can act as a paracrine signal within the immune system, its best known physiological role is within the circulatory (vascular) system, where it acts as a potent vasodilator.  NO, synthesized by NOS in the vascular endothelial cells, diffuses into vascular smooth muscle cells (VSMC) where it binds to the soluble isoform of the enzyme guanylyl cyclase.  This intracellular enzyme, which changes conformation on binding NO, catalyses the conversion of GTP into cyclic guanosine-3’,5’-monophosphate (cGMP) that activates protein kinase G (PKG) to relax the smooth muscle layer around the blood vessels and hence lower blood pressure.