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0
Q

What is a receptor?

A

Molecule that specifically recognises a second molecule (ligand) or family of molecules and in response to ligand binding, brings about regulation of a cellular process.

In the unbound state a receptor is functionally silent.

1
Q

How may chemical signalling be classified?

A

According to their functions:

Hormones (signalling between cells in different tissues via the circulation)

Neurotransmitters (signalling at specialised cell junctions in the nervous system, synapses)

Local chemical mediators (signalling between adjacent cells in the same tissue)

Note: a single molecule may fall into more than one of these categories depending on where it is synthesised and released and its site of action.

2
Q

Describe the affinity of ligand binding at receptor sites

A

The affinity of ligand binding at receptor sites is generally much higher than binding of substrates to enzyme sites.

This is as ligands may only be present in very small concentrations.

3
Q

Give some examples of the role of receptors in cellular physiology

A

Examples include:

Signalling by hormones/local chemical mediators

Neurotransmission

Cellular delivery

Control of gene expression

Cell adhesion

Modulation of the immune response

Sorting of intracellular proteins

Release of intracellular calcium stores

4
Q

What is an Acceptor?

A

Many molecules whose activities are modified by the binding of small chemicals, including drugs, are not strictly receptors.

If their basic function can be carried out without the interaction of a ligand, then they are not, by definition a receptor.

5
Q

What is a ligand? And what are agonists and antagonists?

A

Any molecule that binds specifically to a receptor site.

If the binding of the ligand produces activation of a receptor by changing its conformation in some way, it is an agonist.

If a receptor binds without causing activation on the receptor, it is an antagonist. It opposes the actions of an agonist. Once it binds to receptor, the receptor cannot be bound by an agonist, preventing it from being activated.

6
Q

What is a partial agonist?

A

Agonist which stimulates a receptor but unable to elicit the maximum cell response possible

7
Q

Are Dihydrofolate Reductase and Voltage-Gated Sodium Channels Acceptors or Receptors?

A

Dihydrofolate reductase is inhibited by binding of the drug methotrexate and is sometimes referred to as the methotrexate receptor, this enzyme operates normally in the absence of methotrexate - it is an Acceptor as it operates in absence of ligand and ligand binding alone produces no response.

Voltage - Gated Na+ channels can be modulated by binding of local anaesthetic agents and a variety of neurotoxicity molecules but it opens in the absence of any signalling molecule so it also an acceptor.

8
Q

What happens when the signalling molecule is hydrophilic?

A

The signal recognition site of the receptor must be present on the extracellular face of the cell surface.

Interaction of the signalling molecule with its specific receptor must then result in the activation of a cellular process.

Hence signal transduction is needed as hydrophilic signalling molecules cannot pass through cell membranes.

Signal transduction is brought about by the extracellular receptor at the cell surface transmitting the signal into the cell.

9
Q

What happens when the signalling molecule is hydrophobic?

A

It will be able to pass through the cell membrane (through the lipid bilayer) by diffusion but an intracellular receptor is still required to transduce the signal into a cellular response

e.g. Steroid and thyroid hormones.

10
Q

How can receptors be classified according to the specific physiological signalling molecule (agonist) that they recognise?

A

E.g. Receptor type: nicotinic (agonist: nicotine) and muscarinic (agonist: Muscarine)

Further subclassification is often made on the basis of their ability to be selectively activated by agonist molecules and sub-classification is also often made on the basis of the affinity (a measure of tightness of binding) of a series of antagonists .

11
Q

What are the similarities between receptor binding sites and the active sites and regulatory sites of enzymes?

A

Binding is specific - specificity is governed by shape of the binding cleft in the receptor or enzyme site.

Specificity of binding confers specificity to the regulation of processes which in receptors are involved of the specificity of substrate of an enzyme.

Binding to both receptors and enzyme is most often reversible.

Ligand binding to receptor and regulator binding to enzyme allosteric sites both induce a conformational change and a change in the activity of the molecule (ligand and substrate molecules may also ‘induce a fit’)

There is no chemical modification of ligand in receptor binding sites or enzyme regulatory sites.

12
Q

What are the differences between receptor binding sites and the active sites and regulatory sites of enzymes?

A

The affinity of ligand binding at receptor sites is generally higher than the binding of substrates and regulators to enzyme sites.

The ligand bound to a receptor site is not modified chemically whereas substrate bound in an enzyme active site is modified in a chemical reaction catalysed by the active site.

13
Q

What are the common mechanisms to transduce an extracellular hydrophilic signal into an intracellular event?

A
  1. Membrane-bound receptors with integral ion channels.
  2. Membrane-bound receptors with integral enzyme activity
  3. Membrane- bound receptors which couple to effectors through transducing proteins.
  4. Intracellular receptors for hydrophobic ligands.
14
Q

Describe how Membrane-bound receptors with integral ion channels work

A

Agonist binding to ligand-gated ion channel results in a change in conformation and opening of a gated channel which permits the flow of ions down an electrochemical gradient.

This transduces the signal into an electrical event at the plasma membrane .

15
Q

Describe the subunit structure of the ‘classical’ ligand gated ion channel family

A

They have similar pentameteric subunits.

The subunits have four transmembrane domains e.g. nACh receptors, gamma aminobutyric acid (GABA(A)R), Glycine receptors

One of the four transmembrane domains forms the lining to the channel pore (M2)

Receptors are composed of five homologous, but not identical polypeptide subunits

Agonist binding occurs in the extracellular N-terminal domain of subunits containing a binding site

16
Q

Are there non-classical ligand ion channels?

A

Yes, other structurally distinct ligand-gated ion channel families can also be present in cells e.g ATP-sensitive K+ channels, Ryanodine receptors, IP3 receptor, P2x-purinoceptor (ATP)

17
Q

Describe Membrane-bound receptors with integral enzyme activity

A

Agonist binding to the extracellular domain of these receptors causes a conformational change which activates an intrinsic enzyme activity contained within the protein structure of the receptor e.g. Tyrosine kinase-linked receptors, guanylyl cyclase-linked receptors.

Examples include growth factor receptors such as receptors for insulin, epidermal growth (EGF) and platelet derived growth factor (PDGF) linked directly to tyrosine kinase.

18
Q

Explain Tyrosine Kinase Linked Receptors

A

Binding of ligand to extracellular binding sites activates a protein kinase activity in the cytoplasmic domain of the receptor protein, which autophosphorylates (catalyses the transfer of a phosphate group from ATP on its own structure) tyrosine residues on the cytoplasmic domain of the receptor.

19
Q

What happens to the phosphorylated receptor tyrosine residues?

A

Recognised either by transducing proteins e.g. Insulin receptor substrate-1 (IRS-1), or directly by enzymes containing the phospho tyrosine recognition sites, Src-homology-2 (SH2 domains).

On association with receptor or transducing protein, effector enzymes become activated allosterically or possibly by tyrosine phosphorylation by the receptor kinase, thus transducing the message into an intracellular chemical event.

20
Q

What does a transducer presenting with multiple tyrosine residues mean?

A

The multiple tyrosine residues are phosphorylated after ligand binding and can recruit many different effectors at once.

Activation of 1 receptor –> multiple responses. Often activates a cascade.

21
Q

Describe how membrane-bound receptors which couple to effectors through transducing proteins proteins work

A

No integral enzyme activity or integral ion channels.

Seven transmembrane domain receptors (7TMDR) couple to effector molecules via transducing molecule, a GTP-binding regulatory protein (G-Protein).

This family of receptors is also known as the GPCR family.

Effectors may be enzymes (e.g. Adenylyl cyclase) or ion channels (e.g. Ca2+, K+)

Receptor binding results in a conformational change which activates GDP/GTP exchange in GTP-binding regulatory proteins which transduce the message onto an enzyme or ion channel in the membrane.

22
Q

A wide variety of extracellular signalling molecules utilise specific 7TMD receptors so this structure is common. Give some examples

A

mAChR, dopamine receptors, adrenoceptors, opioid receptors,peptide receptors, light receptors, smell and taste receptors.

Often a number of different types of G-protein receptors exist for a particular agonist e.g. M1-M5 mAChRs

23
Q

What is integrated signalling?

A

Often, separate G-protein coupled receptors will act simultaneously to both stimulate/inhibit the effector.

This is integrated signalling and the two inputs combine to produce a measured effect.

24
Q

Explain how Intracellular receptors for hydrophobic ligands work

A

Hydrophobic ligands (e.g. Steroid hormones, thyroid hormones) pass through the plasma membrane and bind to monomeric receptors in the cytoplasm of nucleus.

In the resting state these receptors are stabilized by association with heat shock or chaperone proteins.

The activated receptor dissociates from the chaperone protein and translocates to the nucleus where it binds to control regions in DNA defined by specific sequences, thereby regulating gene expression.

Compared to extracellular receptors that act through ion channels or enzymes, the action of intracellular receptors is relatively slow as they are dependent on transcription and translation.

25
Q

Explain Amplification in Cellular Signalling

A

The concentration of many extracellular signalling molecules is very low.

For all signalling mechanisms there is the possibility of molecular amplification e.g., by stimulating the activity of an enzyme, the binding of a chemical signal molecule to a single receptor can cause the modification of hundreds or thousands of substrate molecules.

A cascade of such catalytic events can produce further amplification.

26
Q

Cellular Activation and Inhibition depends on the receptor. Explain about cellular activation and inhibition in cardiac pacemaker cells

A

Noradrenaline –> B1-adrenoceptors produces increased Heart Rate

Acetylcholine –> M2 muscarinic receptors produces slowing of Heart Rate

27
Q

Cellular Activation and Inhibition depends on the receptor. Explain about cellular activation and inhibition in hepatocytes

A

Insulin stimulates the synthesis of glycogen from glucose while glucagon stimulates glycogen breakdown to glucose

28
Q

What are the 3 Processes of Membrane Internalisation? - since cell membranes do not become excessively ruffled, there must be an opposite movement of cell membrane back into the cell by endocytosis after release of vesicle contents by exocytosis)

A

Phagocytosis

Pinocytosis

Receptor-mediated Endocytosis

29
Q

Describe Phagocytosis

A

Found only in specialised cells e.g. Macrophages and neutrophils.

In response to binding of a particle to receptors in the plasma membrane, the cell extends pseudopods which permit further receptor interactions and membrane invagination/particle invagination via a membrane-zippering mechanism.

Internalized Phagosomes fuse with lysosomes to fuse phagolysosomes in which the particulate material is degraded.

This process permits the clearance of damaged cellular materials and invading organisms for destruction.

30
Q

Describe Pinocytosis

A

The invagination of the plasma membrane to form a lipid vesicle.

This permits the uptake of impermeable extracellular solutes and retrieval of plasma membrane.

Pinocytosis can be sub-divided into two forms: fluid phase and receptor mediated endocytosis.

31
Q

What is a Endocytosis?

A

The selective internalisation of molecules from extracellular medium into the cell by binding to specific cell surface receptors.

32
Q

Describe Receptor-Mediated Endocytosis (RME)

A

Specific binding of molecules to cell surface receptors permits the selective uptake of substances into the cell.

33
Q

Describe LDLs

A

Low density lipoproteins originate in the liver and consist of a core of cholesterol molecules esterified to fatty acid, a surrounded by a lipid monolayer containing phospholipids, cholesterol and a single protein species, apoproteins B

34
Q

Give an example of receptor mediated endocytosis

A

Cholesterol uptake.

Animal cells requiring cholesterol synthesis, synthesise cell surface LDL-receptors that recognise specially apoprotein B.

Within 10 minutes of binding, the LDL particle is internalised and deliver to the lysosomes where the cholesterol is released from the cholesterol esters.

35
Q

Where are the cell surface LDL-Receptors arranged and located?

A

Localised in clusters over Clathrin Coated Pits that cover ~2% of cell surface.

These pits form spontaneously (association of the coat proteins is energy-independent).

Clathrin spontaneously forms cages - minimum structure is a three-legged triskelion containing Clathrin (heavy chains) and two light chains in the ratio 3:2:1.

It is proposed that the triskelions associate to form a basket-like structure consisting of hexagons and pentagons.

36
Q

What happens when LDL binds to the receptors?

A

The coated pits invaginate to form coated vesicles.

The coated vesicles are quickly uncoated in a process that requires ATP (as they are formed spontaneously).

The ATP-dependent Uncoating protein binds and stabilizes the freed coat proteins.

Clathrin triskelions are recycled back to newly forming Clathrin coated pits.

The uncoated vesicles then fuse with large smooth vesicles called endosomes.

37
Q

Describe the pH inside the endosome

A

The pH of the endosome is maintained between ~5.5-6.0 by an ATP-dependent proton pump.

At this pH the LDL-receptor has low affinity for the LDL particle and the toe dissociate.

Because of this the endosome is also known as COMPARTMENT FOR THE UNCOUPLING OF RECEPTOR AND LIGAND (CURL).

The receptors are sequestered to a domain within the endosome membrane which buds off as a vesicle and recycles the LDL-receptor to the plasma membrane.

38
Q

What happens to the endosomes containing the LDL?

A

They fuse with lysosomes such that the cholesterol can be hydrolysed from the esters and released into the cell.

Thus the LDLs and their receptors are sorted from each other in the endosome.

Ultimately the ligand (LDL) is degraded and the receptor is recycled.

39
Q

How is the Clathrin coat attached to the plasma membrane?

A

By a number of integral membrane adapter proteins which form associations both with the Clathrin and LDL receptors, locating the receptors over the coated pits.

40
Q

Describe the mutations affecting the LDL-receptor in Hypercholesterolaemia

A

3 types of naturally occurring mutations when found in a homozygous individual lead to 3 phenotyoes:

1) receptor deficiency: mutations that prevent expression of the LDL receptor
2) non-functional receptor: no binding of LDL due to a mutation of the LDL binding site on the receptor preventing binding and uptake. Normal coated pits and internalization.
3) receptor binding normal: no internalization due to a deletion in the C-terminal cytoplasmic domain that makes the interaction between the receptor and the Clathrin coat. The LDL receptors will be distributed over the entire cell surface in these patients (instead of in clusters in ~2%)

41
Q

Describe the uptake of Fe3+ irons by Transferrin as an example of RME

A

2 Fe3+ ions bind to Apotransferrin to form Transferrin in the circulation.

Transferrin, but not Apotransferrin, binds to the Transferrin Receptor at neutral pH and is internalised in a similar way to LDL.

On reaching the acidic endosome, the Fe3+ ions are released from the transferrin but at this pH, the Apotransferrin remains associated with the transferrin receptor.

The complex is sorted in the CURL for recycling back to the plasma membrane, where at pH 7.4, the Apotransferrin dissociates from the transferrin receptor again.

Endocytosis of transferrin results in the ligand (transferrin) being recycled and the receptor recycled.

42
Q

Describe the uptake of occupied insulin receptors

A

Most receptors internalized by RME are located over the coated pits but some such as the insulin receptor, only congregate over the coated pits when agonist is bound

. Insulin binding probably induces a conformational change in the insulin receptor that allows it to be recognised by the coated pit.

In the endosome, insulin remains bound to the receptor and the complex is targeted to the lysosomes for degradation.

Endocytosis of insulin results in both the ligand and receptor being degraded.

43
Q

What does the mechanism for the uptake of occupied insulin receptors allow for?

A

Mechanism allows for the reduction of insulin receptors on the membrane surface (down-regulation) which desensitizes the cell to a continued process of high circulating insulin concentration.

44
Q

What is the Cycle of Futility?

A

Basal hyper-insulinaemia means decreased insulin binding as more receptors are degraded leading to insulin resistance (de-sensitised tissue) which leads to decreased tissue response to insulin which leads to increased hepatic glucose produced and decreased cellular glucose uptake which leads to increase hyper-glycemia which leads back to basal hyper-insulinaemia.

45
Q

Describe how receptor mediated endocytosis mode 3 can be a model for the development of Type II diabetes

A

Model for the development of Type 2 diabetes image

46
Q

What is Transcytosis?

A

Some ligands that remain bound to their receptors may be transported across the cell level e.g. maternal immunoglobulins to the foetus via the placenta, transfer of immunoglobulin A (IgA) from the circulation to bile in the liver.

During transport of IgA the receptor is cleaved, resulting in the release of immunoglobulin with a bound ‘secretory component’ derived from the receptor. Endocytosis of immunoglobulin transcytosis results in ligand and receptor being TRANSPORTED.

47
Q

Why are there different modes of Receptor Mediated Endocytosis?

A

Receptors for different ligands enter the cell via the same Clathrin coated pits and the pathway from coated pits to the endosome is common for all proteins that undergo endocytosis.

Different modes of this process can defined on the basis of the destination of internalized receptor and ligand.

Receptors targeted to different cellular destinations, by short amino acid motifs, are sorted within the CURL to discrete regions of the membrane which bud off into transport vesicles.

48
Q

What is Mode 1 of a Receptor Mediated Endocytosis?

A

Fate of Receptor: Recycled

Fate of Ligand: Degraded

Example: LDL

Function: Metabolic Uptake

49
Q

What is Mode 2 of Receptor Mediated Endocytosis?

A

Fate of Receptor: Recycled

Fate of Ligand: Recycled

Example: Transferrin

Function: Metabolic Uptake

50
Q

What is Mode 3 of Receptor Mediated Endocytosis?

A

Fate of Receptor: Degraded

Fate of Ligand: Degraded

Example: Insulin, Epidermal Growth Factor, Immune complexes

Function: Receptor down-regulation (Insulin, Epidermal Growth Factor), Removal from circulation of foreign antigens (Immune complexes)

51
Q

What is Mode 4 of Receptor Mediated Endocytosis?

A

Fate of Receptor: Transported

Fate of Ligand: Transported

Example: Maternal Immunoglobulins, Immunoglobin A

Function: Transport of large molecules across cell e.g. maternal immunity to foetus via placenta, transport of IgA from circulation to bile in liver

52
Q

How do membrane-enveloped viruses and some toxins take advantage of Receptor-Mediated-Endocytosis to gain entry into the cell?

A

They exploit endocytic pathways to enter cells after adventitious binding to receptors in the plasma membrane.

They bind to cells by fortuitous association with cell receptors.

Enter cells via Clathrin-coated pits (RME).

Once in the endosome the acidic pH leads to the unfolding of hydrophobic domains in membrane fusion proteins.

Inserting membrane fusion proteins into the endosome membrane leads to membrane fusion and to release the viral RNA into the cell cytoplasm where it can be translated and replicated by the host cell’s machinery to form new viral toxins

New viruses are released by budding off the cell membrane.

53
Q

Give examples of entry of toxins by receptor-mediated endocytosis?

A

Cholera toxin and Diptheria toxin both bind to GM1 Ganglioside.

54
Q

Describe the importantance of calmodulin in smooth muscle

A
  • Troponin is absent in smooth muscle.
  • Instead Ca2+ sensitivity is mediated by calmodulin which binds and activates myosin light chain kinase, leading to phosphorylation of myosin light chains.
  • Smooth muscle myosin head groups bind to actin filaments only when the light chains are in the phosphorylated form.
  • On restoration of intracellular [Ca2+] the calmodulin dissociates from MLCK, rendering it inactive
  • Relaxation of smooth muscle follows when a myosin light chain phosphatase returns the myosin filament to the inactive form
55
Q

How is the release of sarcoplasmic reticulum stores Ca2+ coupled to membrane depolarization in skeletal muscle?

A
  • The T Tubular system ensures that the action potential stimulus for contraction is transmitted rapidly deep within the fibre.
  • The L-Type Voltage-Sensitive Ca2+ channels in the T-Tubule System change conformation in response to the depolarization.
  • They are physically coupled to Ryanodine-sensitive Ca2+ Channels in the SR and the L-type channels stimulate the Ryanodine-sensitive channels to open and release Ca2+ from the SR store.
  • SERCA returns Ca2+ to the store
56
Q

Give FOUR membrane-bound receptors with integral enzyme activity

A

Atrial natriuretic peptide (ANP) receptor

Epidermal growth factor (EGF) receptor

Insulin receptor

Platelet-derived growth factor (PDGF) receptor