S377 Chapter 13

Sometimes cells communicate directly through [blank_start]gap junctions[blank_end], which connects the [blank_start]cytoplasm[blank_end] of the cells and allows small molecules to travel through. This is different from normal signalling. [blank_start]Contact-dependent[blank_end] signalling involves signalling molecules not being [blank_start]secreted[blank_end], but expressed on the [blank_start]plasma membrane[blank_end] of the cell, and picked up by [blank_start]receptors[blank_end] on the plasma membrane of another cell. For example, when [blank_start]antigens[blank_end] are presented to [blank_start]T cells[blank_end] in the [blank_start]immune system[blank_end].

Label the diagram

[blank_start]Paracrine signalling[blank_end] affects local cells. Cells closer to the source react differently to those further away due to [blank_start]concentration gradients[blank_end]. Signalling molecules used are either rapidly taken up by target cells or degraded by [blank_start]extracellular enzymes[blank_end] to keep the effects limited to a small area. [blank_start]Autocrine signalling[blank_end] is an oddity, and may occur in [blank_start]tumour cells[blank_end] to allow them to keep growing inappropriately. Also happens in [blank_start]development[blank_end]. [blank_start]Endocrine signalling[blank_end] involves signalling cells very far away, with the signalling molecules often transported in the [blank_start]bloodstream[blank_end]. These signalling molecules are called [blank_start]hormones[blank_end] (though there is lots of variation) and are not [blank_start]water soluble[blank_end] so last longer than [blank_start]paracrine signalling molecules[blank_end]. [blank_start]Electrical signalling[blank_end] involves neurons, where the signal is transmitted electrically along [blank_start]axons[blank_end] before being turned into a chemical signal at [blank_start]synapses[blank_end], to cross the [blank_start]synaptic cleft[blank_end]. The chemicals signalling molecules are called [blank_start]neurotransmitters[blank_end] (these are also very varied).

Label the 4 main classes of receptors

In a basic model of [blank_start]signal transduction[blank_end], a signalling molecule binds to a [blank_start]specific receptor[blank_end], and this activates a sequence (or web) of [blank_start]intracellular signalling molecules[blank_end] that spread the information to relevant parts of the cell, activating [blank_start]target molecules[blank_end], which effect a [blank_start]cellular response[blank_end].

Signalling between cells can be contact dependent or via secreted signalling molecules. The latter comprise p[blank_start]aracrine[blank_end], a[blank_start]utocrine[blank_end], e[blank_start]ndocrine[blank_end] or electrical signalling.

There are four types of cell surface receptors: ion [blank_start]channel[blank_end] receptors, 7-[blank_start]helix transmembrane[blank_end] receptors, receptors with [blank_start]intrinsic enzymatic[blank_end] activity, and enzyme-associated ([blank_start]recruiter[blank_end]) receptors. Receptors with intrinsic [blank_start]transcriptional[blank_end] activity are mostly [blank_start]intracellular[blank_end].

Two basic categories of signalling molecules intervene in signal transduction, according to the spatial and temporal requirements of the signalling pathway. (a) Small diffusible signalling molecules (‘[blank_start]second messengers[blank_end]’) enable rapid signal [blank_start]amplification[blank_end] and a [blank_start]widespread[blank_end] cellular response. (b) Signalling [blank_start]proteins[blank_end] fulfil many roles (by virtue of protein–protein interaction and protein regulation), including signal [blank_start]integration[blank_end], modulation, t[blank_start]ransduction[blank_end] and a[blank_start]nchoring[blank_end] functions.

[blank_start]G proteins[blank_end] and proteins activated by [blank_start]phosphorylation[blank_end] on tyrosine, serine and/or threonine residues can act as [blank_start]molecular switches[blank_end].

The [blank_start]subcellular location[blank_end] of the signalling protein is critical to its [blank_start]function[blank_end], and this is aided by [blank_start]transient[blank_end] or preassembled [blank_start]signalling complexes[blank_end].

Specific binding of [blank_start]signalling proteins[blank_end] to each other is critical for the effective [blank_start]transduction[blank_end] of the signal. [blank_start]Binding domains[blank_end] allow transient binding to [blank_start]specific[blank_end] (often phosphorylated) amino acid sequences or to [blank_start]phospholipids[blank_end].

The dissociation constant (KD) describes the affinity between receptors and their ligands. (D is subscript!) The higher the value of KD , the [blank_start]lower[blank_end] the affinity of a receptor for its ligand.

Ligands are classed as agonists or antagonists. [blank_start]Agonists[blank_end] usually work by binding to the ligand binding site and [blank_start]promoting[blank_end] its [blank_start]active conformation[blank_end]. [blank_start]Antagonists[blank_end] bind to the receptor, but [blank_start]do not promote[blank_end] the switch to the active conformation. A single receptor may be able to bind several [blank_start]different ligands[blank_end], and a single ligand may be able to bind to [blank_start]several receptors[blank_end].

[blank_start]Receptors[blank_end] comprise a limited number of [blank_start]structural motifs[blank_end], which determine [blank_start]binding affinity[blank_end] and specificity of receptor–ligand complexes. Some ligands bind to [blank_start]several receptors[blank_end] and some receptors bind to [blank_start]several ligands[blank_end].

[blank_start]Acetylcholine[blank_end] is a good example of a ligand with two structurally different kinds of [blank_start]receptor[blank_end]. [blank_start]Nicotinic[blank_end] receptors are ion channels, which are found predominantly in [blank_start]skeletal muscle[blank_end], and are stimulated by nicotine. Nicotinic receptor [blank_start]antagonists[blank_end] include the toxins α -bungarotoxin and tubocurarine. Acetylcholine binds at two sites within the channel. [blank_start]Muscarinic[blank_end] receptors, in contrast, are [blank_start]7TM G protein-coupled receptors[blank_end], found (for example) in [blank_start]cardiac muscle[blank_end]. Muscarine acts as an [blank_start]agonist[blank_end], whereas [blank_start]atropine[blank_end] acts as an antagonist. Acetylcholine binds in the core region of the transmembrane helical segments.

[blank_start]Adrenalin[blank_end] has a range of structurally related [blank_start]7TM G protein-coupled receptors[blank_end], with different tissue distributions and different [blank_start]affinities[blank_end] for numerous agonists and antagonists. Adrenalin, like acetylcholine, binds in the [blank_start]core region[blank_end] of the receptor, though other GPCRs can be activated in a variety of ways.

Mechanisms for [blank_start]receptor activation[blank_end] are varied and include [blank_start]conformational[blank_end] changes (ion-channel receptors and 7TM), [blank_start]homo- or heterodimerization[blank_end] (receptors with intrinsic enzymatic activity and recruiter receptors) or even proteolysis.

For most [blank_start]7TM receptors[blank_end] (the exception being the Frizzled class of 7TM receptors), [blank_start]conformational change[blank_end] on ligand binding activates associated [blank_start]cytoplasmic G proteins[blank_end]. Hence, they are called ‘[blank_start]G protein-coupled receptors[blank_end]’.

Receptors with [blank_start]intrinsic enzymatic activity[blank_end] include [blank_start]receptor tyrosine kinases[blank_end], receptor serine–threonine kinases, receptor tyrosine phosphatases, and receptor guanylyl cyclases. Most RTKs are activated by [blank_start]dimerization[blank_end] on ligand binding, leading to [blank_start]autophosphorylation[blank_end] of the cytoplasmic portion of the receptor. [blank_start]Phosphorylated[blank_end] tyrosine residues serve as docking sites for SH2-containing signalling proteins, which also recognize sequence-specific flanking motifs.

[blank_start]Dimerization[blank_end] of [blank_start]recruiter receptors[blank_end] facilitates the interaction between the [blank_start]membrane-bound[blank_end] receptor and [blank_start]cytosolic proteins[blank_end] with intrinsic enzymatic activity such as [blank_start]kinases[blank_end].

Receptors can be [blank_start]inactivated[blank_end] by [blank_start]removal[blank_end] of the [blank_start]ligand[blank_end], or by receptor [blank_start]desensitization[blank_end], which can be by i[blank_start]nactivation[blank_end], by s[blank_start]equestration[blank_end] or by d[blank_start]egradation[blank_end] of the receptor.

Some [blank_start]signalling molecules[blank_end] can diffuse across the [blank_start]plasma membrane[blank_end], and so have [blank_start]intracellular[blank_end], rather than cell surface receptors. Small hydrophobic ligands such as [blank_start]steroid hormones[blank_end] bind to members of the [blank_start]nuclear receptor group[blank_end], which undergo [blank_start]conformational change[blank_end] and bind to specific [blank_start]DNA sequences[blank_end], stimulating [blank_start]transcription[blank_end] of target genes.

Label the diagram of signalling through G protein-coupled receptors (GPCRs)

Label the diagram of one type of signalling involving cAMP

[blank_start]Heterotrimeric G proteins[blank_end] are tethered to the [blank_start]internal surface[blank_end] of the plasma membrane, and are activated by [blank_start]conformational change[blank_end] within [blank_start]7TM receptors[blank_end]. There are many different [blank_start]α subunits[blank_end] (and a few βγ subunits), which interact with [blank_start]different receptors[blank_end] and different [blank_start]effectors[blank_end]. The major targets of G proteins include ion [blank_start]channels[blank_end], [blank_start]adenylyl cyclase[blank_end] ([blank_start]activated[blank_end] by Gαs and [blank_start]inhibited[blank_end] by Gαi) and PLC- β (activated by Gαq).

[blank_start]Phosphatidylinositol (PI)[blank_end] is the precursor of a family of small lipid [blank_start]second messengers[blank_end]. The inositol ring can be further [blank_start]phosphorylated[blank_end] at positions 3, 4, and 5 by lipid kinases. [blank_start]PI 3-kinase[blank_end] specializes in phosphorylating the hydroxyl group at the [blank_start]3 position[blank_end], thus generating the active signalling molecules PI(3,4)P2 and PI(3,4,5)P3 . The [blank_start]phosphorylated 3 position[blank_end] is recognized as a docking site by [blank_start]PH domain-containing proteins[blank_end], thus providing a mechanism for signalling proteins to be recruited to the membrane.

[blank_start]Phospholipase C enzymes[blank_end] (especially PLC- β , activated by [blank_start]G proteins[blank_end], and [blank_start]RTK-activated[blank_end] PLC- γ ) cleave PI(4,5)P2 to generate [blank_start]diacylglycerol (DAG)[blank_end] and inositol 1,4,5-triphosphate (IP3 ). DAG remains embedded in the [blank_start]membrane[blank_end], where it activates [blank_start]protein kinase C (PKC)[blank_end]. [blank_start]IP3[blank_end] diffuses through the cytosol, and opens [blank_start]IP3 -gated calcium channels[blank_end], releasing [blank_start]stored calcium[blank_end] into the cytosol.

The [blank_start]Ca2+ ion[blank_end] is an important [blank_start]second messenger[blank_end], which enters the cytosol from the [blank_start]extracellular space[blank_end] through specific channels on the plasma membrane, or is rapidly released from [blank_start]stores[blank_end] into the cytoplasm. Calcium channels include [blank_start]IP3 -gated[blank_end] calcium channels, [blank_start]voltage-[blank_end]dependent calcium channels, or ryanodine receptors in skeletal muscle cells. It activates numerous Ca2+ -dependent proteins, [blank_start]including PKC[blank_end], but many of its effects are mediated via [blank_start]calmodulin[blank_end], which has four allosteric Ca2+ binding sites. Ca2+ /calmodulin then binds to and regulates [blank_start]target proteins[blank_end], especially Ca2+ /calmodulin-dependent protein kinases ([blank_start]CaM kinases[blank_end]).

[blank_start]Cyclic AMP (cAMP)[blank_end] is another important [blank_start]second messenger[blank_end], synthesized from [blank_start]ATP[blank_end] by [blank_start]adenylyl cyclase[blank_end] (which is activated or [blank_start]inhibited[blank_end] by different G protein subtypes). It can open cAMP-gated [blank_start]ion channels[blank_end], but it mediates many of its effects through cAMP-dependent protein kinase A ([blank_start]PKA[blank_end]), whose roles include regulating [blank_start]glycogen[blank_end] metabolism, and [blank_start]phosphorylation[blank_end] of a transcription factor ([blank_start]CREB[blank_end]) that binds to the [blank_start]cAMP response element (CRE)[blank_end].

[blank_start]Cyclic GMP[blank_end] is synthesized by guanylyl cyclase. Its targets include [blank_start]cGMP-gated[blank_end] ion channels and a [blank_start]cGMP-dependent kinase (PKG)[blank_end].

[blank_start]Ras[blank_end] is the archetypal [blank_start]monomeric[blank_end], or small, [blank_start]G protein[blank_end]. Ras classically operates downstream of [blank_start]growth factor receptors[blank_end]: Grb-2, an SH2/SH3-containing adaptor protein, binds to phosphotyrosines on the activated RTK, and recruits Sos to the membrane environment; Sos promotes GTP binding by Ras. [blank_start]Activated Ras[blank_end] has more than one target, including [blank_start]PI 3-kinase[blank_end], but its most important downstream pathway is the [blank_start]MAP kinase pathway[blank_end]. Activated Ras recruits [blank_start]Raf[blank_end] to the membrane, where it is activated and then [blank_start]phosphorylates[blank_end] MEK, which then phosphorylates ERK, a [blank_start]MAP kinase[blank_end]. These have multiple cytoplasmic and [blank_start]transcription factor[blank_end] targets involved in cell [blank_start]growth and division[blank_end] or differentiation.

[blank_start]Protein kinase families[blank_end] involved at various points in signalling pathways include [blank_start]receptor tyrosine kinases[blank_end] (for example, the EGF receptor), non-receptor [blank_start]tyrosine kinases[blank_end] (such as Src and JAK), serine[blank_start]–threonine kinases[blank_end] such as PKC, PKA, MAP kinases and the TGF receptor, and rare [blank_start]dual-specificity kinases[blank_end] such as MEK.

[blank_start]Protein phosphatases[blank_end] dephosphorylate proteins, and are grouped according to their targets, as are protein kinases. They include protein tyrosine [blank_start]phosphatases[blank_end], [blank_start]serine–threonine[blank_end] phosphatases, and a few dual-specificity phosphatases.

The duration of ERK activity determines [blank_start]activation[blank_end] of different [blank_start]transcription factors[blank_end] (SRF/TCF for immediate early genes; AP-1 for other target genes).

Label the signalling pathways

Label the diagram

Label the diagram

Label the MAP kinase pathway

Label the JAK-STAT pathway

[blank_start]Glycogen[blank_end] metabolism is controlled by two enzymes, glycogen [blank_start]synthase[blank_end] (mediating glycogen synthesis) and [blank_start]phosphorylase[blank_end] (mediating glycogen breakdown).

[blank_start]Three[blank_end] pathways converge in the regulation of [blank_start]glycogen synthase[blank_end]: cAMP/PKA and GSK-3β are [blank_start]negative[blank_end] regulators, whereas ISPK/PP1G [blank_start]positively[blank_end] regulate the activity of glycogen synthase.

[blank_start]Insulin[blank_end] and adrenalin have opposite effects on glycogen [blank_start]synthesis[blank_end]: insulin [blank_start]promotes[blank_end] glycogen synthesis by activating ISPK/PP1G and by inhibiting GSK-3β by the [blank_start]PI3K/PKB[blank_end] pathway, whereas [blank_start]adrenalin[blank_end] inhibits glycogen synthase by the [blank_start]cAMP/PKA[blank_end] pathway.

Three pathways converge in the activation of [blank_start]phosphorylase[blank_end] by phosphorylase [blank_start]kinase[blank_end]: Ca2+ and [blank_start]PKA[blank_end] activate phosphorylase kinase, whereas [blank_start]PP1[blank_end] is a negative regulator.

[blank_start]Acetylcholine[blank_end], adrenalin and [blank_start]glucagon[blank_end] promote [blank_start]glycogen[blank_end] breakdown, whereas [blank_start]insulin[blank_end] inhibits it.

[blank_start]Acetylcholine[blank_end] in skeletal muscle and [blank_start]adrenalin[blank_end] in liver activate [blank_start]phosphorylase[blank_end] kinase by a common mechanism, an increase in cytosolic [blank_start]Ca2+[blank_end] , although the effect of ACh is by [blank_start]voltage-dependent[blank_end] channels and that of adrenalin by [blank_start]IP3-gated Ca2+[blank_end] channels.

[blank_start]Adrenalin[blank_end] in muscle and [blank_start]glucagon[blank_end] in liver activate [blank_start]phosphorylase[blank_end] kinase by a common mechanism, namely an increase in [blank_start]cytosolic cAMP[blank_end] and subsequent activation of [blank_start]PKA[blank_end]. In addition, PKA further activates phosphorylase kinase in skeletal muscle by inhibition of [blank_start]PP1[blank_end] either directly or indirectly.

[blank_start]Insulin[blank_end] has an opposite effect to [blank_start]adrenalin[blank_end] on [blank_start]glycogen[blank_end] breakdown, namely the [blank_start]inhibition[blank_end] of glycogen breakdown by [blank_start]activation[blank_end] of the phosphorylase kinase inhibitor, [blank_start]PP1[blank_end].

Activated [blank_start]PI 3-kinase[blank_end] activates [blank_start]PKB/Akt[blank_end], which exerts a number of anti-apoptotic actions by [blank_start]phosphorylation[blank_end] of downstream proteins: (1) it phosphorylates [blank_start]Bad[blank_end], thereby promoting Bad’s [blank_start]sequestration[blank_end] by an adaptor protein (not shown); (2) it phosphorylates and thereby [blank_start]inhibits caspase 9[blank_end] (in humans); (3) it phosphorylates and thereby [blank_start]inhibits[blank_end] members of the Forkhead family of [blank_start]transcription factors[blank_end], which stimulate transcription of [blank_start]pro-apoptotic genes[blank_end]; and (4) it phosphorylates and thereby [blank_start]activates[blank_end] the transcription factor [blank_start]CREB[blank_end], which stimulates transcription of [blank_start]anti-apoptotic genes[blank_end].