Physiology of Membrane Receptors

Question 1

__________________ are proteins with high affinity to a particular ligand, usually a hormone or neurotransmitter, other proteins, light or other chemicals.

Answer 1

Receptors

[The attachment of a ligand to its receptor will cause a conformational change which will stimulate an intracellular event, which will then transduce the effect of the bound ligand.]

Question 2

(a) Membrane receptors are important for ________________ signals.
(b) The ligand-binding domain faces the ____(i)____ of a cell, while the coupling or effector domain faces the ____(ii)____.

Answer 2

(a) hydrophilic
(b) (i) exterior (ii) interior or cytosolic side

Question 3

Most cell-surface membrane receptor proteins belong to one of four classes, defined by the transduction mechanism. List these classes.

Answer 3

1. ion-channel-linked receptors
2. G-protein-coupled receptors
3. enzyme-linked receptor
4. tyrosine receptor kinases

Question 4

Ligand-gated ion channels are also called _________________ receptors.

Answer 4

ionotropic

NB: Ionotropic receptors (sometimes called fast receptors) are receptors that include an ion channel. Examples of ionotropic receptors include receptors for serotonin (5-HT3 [5-hydroxytryptamine 3] receptors) and acetylcholine (nicotinic receptors).

Question 5

The opening and closing of signal-gated ion channels are controlled by ________________ receptors.

Answer 5

metabotropic

NB: Metabotropic receptors (sometimes called slow receptors) are GPCRs (G-protein coupled receptors) that do not have an ion channel but use the G-protein to bind to an ion channel intracellularly causing its opening. [Note that it can be the βγ-dimer of the activated G-protein binding to a K+ channel intracellularly causing its opening or the α-stimulatory subunit of the G-protein binding to and activating adenylyl cyclase hence the enzyme converts ATP to cAMP, then cAMP activates a cAMP-dependent kinase that phosphorylates an effector protein, and that protein binds to an ion channel intracellularly causing it to open.]

Question 6

Discuss GPCRs.

Answer 6

⚚ All the GPCRs that have been characterized to date are proteins that span the cell membrane seven times. Because of this structure they are alternatively referred to as seven-helix receptors or serpentine receptors.
⚚ The N-terminus is extracellular, the C-terminus is intracellular.
⚚ The segment of the GPCR that interacts with G-protein is intracellular and in between the 5th and 6th transmembrane domains.
⚚ These receptors assemble into a barrel-like structure.
⚚ Upon ligand binding, a conformational change activates a resting heterotrimeric G-protein associated with the cytoplasmic leaf of the plasma membrane.
⚚ Activation of a single receptor can result in 1, 10, or more active heterotrimeric G-proteins, providing amplification as well as transduction of the first messenger.
⚚ Activation of the GPCR is through phosphorylation of the cytoplasmic side of the receptor after the ligand binds onto the receptor.
⚚ Because of their diversity and importance in cellular signaling pathways, GPCRs are prime targets for drug discovery.

Question 7

Which two principal signal transduction pathways involve the GPCRs?

Answer 7

(a) the cAMP signal pathway
(b) phosphatidylinositol signal pathway

Question 8

Give examples of ligands for G-protein- coupled receptors.

Answer 8

Neurotransmitters:
Epinephrine, Norepinephrine, Dopamine, 5-Hydroxytryptamine, Histamine, Acetylcholine, Adenosine, Opioids

Tachykinins:
Substance P, Neurokinin A, Neuropeptide K

Other peptides:
Angiotensin II, Arginine vasopressin, Oxytocin, VIP, GRP, TRH, PTH

Glycoprotein hormones:
TSH, FSH, LH, hCG

Arachidonic acid derivatives:
Thromboxane A2

Other:
Odorants, Tastants, Endothelins, Platelet-activating factor, Cannabinoids, Light, Cations (eg, Ca2+, Mg2+)

Question 9

Make notes on enzyme-linked receptors.

Answer 9

⚛︎ Either function directly as enzymes or are directly associated with enzymes that they activate.
⚛︎ They are formed by single-pass transmembrane proteins that have their ligand-binding site outside the cell and their catalytic or enzyme-binding site inside.
⚛︎ Enzyme-linked receptors are mainly protein kinases, or are associated with protein kinases, and ligand binding to them causes the phosphorylation of specific sets of proteins in the target cell.
Tyrosine kinase associated receptors e.g. those in the JAK family are examples.

Question 10

Describe the mechanism of action of RTKs (receptor tyrosine kinases).

Answer 10

(1) Binding of a ligand (e.g., EGF, platelet-derived growth factor, insulin, FGF) to the extracellular domain of a specific RTK leads to dimerization of two receptor monomers to activate the receptor.
(2) The activated RTK undergoes autophosphorylation of itself on tyrosine residues in the cytoplasmic domain.
(3) Autophosphorylation activates the tyrosine kinase domain (TKD) of the receptor.
(4) The activated RTK initiates downstream signaling cascades.

Note:
Ras is a small GTP-binding protein different from the heterotrimeric G proteins activated by GPCR.

Question 11

Outline a cascade of events related to activation of tyrosine kinase-associated receptors.

Answer 11

Receptors for cytokines and colony-stimulating factors differ from the other growth factors in that most of them do not have tyrosine kinase domains in their cytoplasmic portions and some have little or no cytoplasmic tail. However, they initiate tyrosine kinase activity in the cytoplasm. In particular, they activate the so-called Janus tyrosine kinases (JAKs) in the cytoplasm. These in turn phosphorylate signal transducer and activator of transcription (STAT) proteins. The phosphorylated STATs form homodimers and heterodimers and move to the nucleus, where they act as transcription factors. There are four known mammalian JAKs and seven known STATs.
In summary the events can occur as follows: 1) A Ligand binding leads to dimerization of receptor 2) Receptor dimerization leads to its activation and tyrosine phosphorylation of JAKs 3) JAKs phosphorylate STATs 4) STATs dimerize and move to nucleus, where they bind to response elements on DNA where they cause gene transcription.

Question 12

When membrane receptors bind specific messenger molecules on the exterior surface of the cell, either of two types of response may occur, which are?

Answer 12

1. direct response e.g. Ach receptor of nervous tissue
2. second messenger involvement

Question 13

State the properties of membrane receptors.

Answer 13

1. The bound signal (1st messenger) induces a conformational change in the receptor that is transmitted through the membrane-anchoring domain onto the coupling domain localized at the inner face of the cell membrane.
2. A variety of messengers can bind to various tissues giving either positive or negative responses depending on the type of receptor.
3. Saturability: there are a finite number of receptors available, but the messenger concentration can continue increasing…now very low concentrations of messengers may give a large response.
4. Cell surface receptors are integral membrane proteins with three basic domains: extracellular domains, transmembrane domains, intracellular domains.
5. Distinctive variations in receptor structure have been identified.
6. Turnover of receptors and other membrane proteins is dependent on endocytosis or ubiquitin-mediated protein degradation via the proteasome pathway.
7. Competition: is the ability of molecules very similar in structure to the normal messenger to combine with the receptor and thereby prevent the normal messenger from doing so.
   [Q. What are agonists, inverse agonists and antagonists?]
8. Down-regulation: Receptors are subject to physiological regulation and so can be decreased as desired.
9. Up-regulation: In this case cells exposed chronically to very low concentration of messenger may develop many more receptors for that messenger, thereby becoming supersensitive.

Question 14

State the methods of receptor regulation.

Answer 14

⚛︎ Alteration in the number of receptors:
Upregulation: Increased expression of receptor molecules on the cell surface due to prolonged deprivation of interaction with their physiological neurotransmitter.
Downregulation: Decreased expression of receptors when the signal is strong.
⚛︎ Alteration in location
Receptors can be internalized (endocytosis) or redistributed within the cell, impacting their availability for ligand binding.
⚛︎ Alteration in affinity
Receptor affinity for ligands can change due to various factors, affecting their responsiveness.

Question 15

How does cholera toxin stimulate the activity of the adenylyl cyclase system?

Answer 15

Inhibits the GTPase activity of the alpha subunit, creating a continually active Gs.

Question 16

How does pertussis toxin stimulate the activity of the adenylyl cyclase system?

Answer 16

Prevents the dissociation of GDP from the nucleotide binding site, creating a continually inactive Gi

Question 17

What are G proteins?

Answer 17

G-Proteins (Guanine nucleotide-binding proteins) are molecular switches involved in transmitting signals from various stimuli outside the cell to its interior.
They act as transducers in signaling pathways, converting external signals into cellular responses.

Question 18

Briefly discuss the structure of G-proteins.

Answer 18

Typically composed of three subunits: alpha (α), beta (β), and gamma (γ).
The α-subunit binds guanine nucleotides (GDP and GTP) and has intrinsic GTPase activity.
The β and γ subunits usually function as a stable dimer.

Question 19

Discuss G-proteins in terms of activation and deactivation.

Answer 19

Activation Mechanism:
Inactive State: Bound to GDP.
Active State: Binding to GTP, often facilitated by a receptor (G-protein-coupled receptor or GPCR) after ligand binding.
GTP-bound α-subunit dissociates from the βγ dimer, both of which can activate downstream signaling pathways.

Deactivation:
The intrinsic GTPase activity of the α-subunit hydrolyzes GTP to GDP, returning the G-protein to its inactive state.

Question 20

Briefly describe the signalling pathways of G-proteins. [GPCR pathway and second messengers] Also state the functional roles of these pathways.

Answer 20

GPCR Pathway: Involves G-proteins in transmitting signals from receptors to effector proteins like adenylate cyclase or phospholipase C.
Second Messengers: Activation of effectors leads to production of second messengers (e.g., cAMP, IP3) which amplify the signal.

Functional roles:
✓ Regulation of metabolic enzymes, ion channels, and other cell signaling pathways.
✓ Involved in sensory perception, immune responses, and cell growth

Question 21

What is the clinical relevance of G-proteins.

Answer 21

Mutations and dysregulation in G-proteins are implicated in various diseases, including cancer (e.g., mutated Ras), and some inherited disorders.
Targeted by many drugs that modulate GPCR activity, impacting therapeutic areas like cardiovascular disease and mental health.

Question 22

Give examples of types of G-proteins as well as their functions.

Answer 22

Gs Proteins: Stimulate adenylate cyclase, increasing cAMP levels.
Gi Proteins: Inhibit adenylate cyclase, reducing cAMP levels.
Gq Proteins: Activate phospholipase C, generating IP3 and DAG as second messengers.
Ras Proteins: Involved in growth factor signaling and oncogenic transformation.

Question 23

Using your knowledge of G proteins, explain how Cholera toxin causes diarrhea.

Answer 23

Cholera toxin ADP-ribosylates the alpha subunits of Gs’s and abolishes the GTPase activity of the subunit, creating a continually active Gs. Thus, the effect of the toxin is to stimulate the activity of the adenylyl cyclase system in target cells.

Question 24

Using your knowledge of G proteins, explain how Pertussis toxin causes whooping cough.

Answer 24

Pertussis toxin ADP-ribosylates the alpha-subunits of Gi’s and prevents the dissociation of GDP form the nucleotide binding site, creating a continually inactive Gi. Thus, the effect of the toxin is to stimulate the activity of the adenylyl cyclase system in target cells.

Question 25

Discuss G-protein as a molecular switch.