PDA IB Lecture 6, 7, & 8: Receptors and Signal Transduction

Drug + Receptor ---> Drug-receptor Complex ---> _______________

Altered function

A drug is useful if....

it has a beneficial therapeutic purpose and offers an acceptable balance between wanted and unwanted effects

Drugs are potentially capable of changing any function by.....

modulating the rates of ongoing process

- they do NOT add new functions

T/F: Drugs may potentially add new functions

FALSE

they do NOT add new functions

The 2 different groups drug targets can be classified into:

1. The usual macromolecules without endogenous ligand

2. "Real receptors". receptors for endogenous regulatory ligands

1. The usual macromolecules without endogenous ligand examples (5)

- receptors for these drugs are enzymes, transporters, channels, proteins, nucleic acids, etc.

- none are real physiological receptors

2. "Real receptors", receptors for endogenous regulatory ligands exmaples (4)

hormones, neurotransmitters, growth factors, cytokines

- sense the presence of these ligands and initiate responses.

Drug-receptor properties: Saturability

maximum saturation occurring with occupation of all the sites of a receptor (ligand binding domains)

Drug-receptor properties: Selectivity

the extent to which a receptor binds with a particular drug rather than other molecules

Selectivity depends both on the receptor and on the _______, _________, and ______________________ of the drug molecule

size, shape, and bioelectrical charge

Drug-receptor properties: Reversibility

the drug/receptor binding/interaction is generally not permanent (some drugs may covalently bind to receptor, resulting in permanent)

Drug-receptor properties: Affinity

how avidly or "tightly" a receptor binds a given drug molecule

(High affinity for a drug means a lower drug concentration needed for full saturation)

Drug-receptor properties: Efficacy

the size of the intracellular or drug effect when the drug and receptor interact

Antagonists*****

- zero efficacy drugs

- generally, have no effect other than preventing the receptor from being activated by an agonist drug

Full agonist*******

drug has high efficacy and can produce the maximum effect on receptors at a sufficient concentration

Partial agonist***** (aka inverse agonist)

drugs have a lower efficacy and cannot produce the maximal effect at any drug concentration level

Allosteric**** regulation of recetpors

- receptors with multiple binding sites

- allosteric binding sites are usually sites other than that for the endogenous agonist

- activation of these sites can change the shape of the receptor, positively or negatively regulating its affinity for the primary endogenous ligand

the 2 functions of receptors****

- sensing the ligand (binding)

- transmitting the message into cell

Most signal-transducing receptors are integral components of the cellular membrane: (2)

-what does this orientation permit?

- extracellular ligand binding domain

- intracellular signal transmitting domain

- permits chemical diversity in signaling molecules

Several signal-transducing receptors are found in the....

Most are present in ___________ numbers

cytoplasm or nucleus

- present in small numbers (few thousand per cell)

T/F: There are often many isoforms of an "individual" receptor

TRUE

(different isoforms may have different mechanism of signal transduction- regulatory flexibility)

4 types of receptors

- Ion channels (ligand gated, voltage gated)

- G-Protein Coupled Receptors (aka seven-(pass)-transmembrane domain receptors)

- Receptor tyrosine kinase

- Intracellular hormone receptors

Neurotransmitter

what are they generated by? to do what?

chemical messengers that are generated by the nerve cell to transmit the message from the cell across the synapse to a target cell

What can the target of a neurotransmitter be? (3)

another nerve cell, a muscle cell, or a gland cell

What are neurotransmitters released from? Where are they released into?

What are they received by?

- released from synaptic vesicles in synapses

- released into the synaptic cleft

- received by neurotransmitter receptors on target cell

Neurotransmitters are classified into 2 categories:

Metabotropic and ionotropic receptors

Fast neurotransmission (ionotropic receptors) (2)

- responses consist of an _________________ and tend to be ______________

- are their effect direct or indirect? does it require multiple steps?

- Responses to activation of receptors consisting of an ion channel tend to be rapid (milliseconds)

- effects are direct and generally do not require multiple steps leading to second-messenger generation and activation of a signaling pathway

Slow Neurotransmission (metabotropic receptors) (5)

- is it faster or slower?

- mediated by......

- what do they consist of?

- Slower transmission (still fast, seconds)

- mediated by neurotransmitters that do not bind to ion channels (effectors and second messengers) but to receptors with very different architecture, metabotropic receptors

- Upon activation, these receptors generate second messengers

- consist of GPCRs

- other metabotropic receptors include tyrosine kinases and guanylyl cyclase receptors

Ionotropic receptors (Ion channels) are needed to establish the.....

the electrochemical gradients required to maintain membrane potential

All cells express ion transporters for.....(4)

Na+, K+, Ca2+, and Cl-

What are passive ion fluxes down cellular electrochemical gradients regulated by?

a large family of ion channels located in the membrane

Ionotropic receptors are classified as 2 groups:

- Voltage-gated ion channel

- Ligand-gated ion channel

Voltage-gated ion channels

multisubunit protein complexes that respond to changes in membrane potential with conformational changes

- very fast response (milliseconds to seconds)

(leading to opening/closing)

(isoforms for Na+, K+, Ca2+, and Cl- ions)

*******The sodium and potassium voltage-gated channel play crucial roles in....

regulating physiological function of nerves and muscle

********The voltage-gated calcium channels play crucial roles in...

neurotransmitter release in pre-synaptic nerve ending

Differences in the concentrations of ions on opposite sides of a cellular membrane lead to...

a voltage called the membrane potential

- typical values of membrane potential are in the range -40 mV to -70 mV

What is the voltage sensing portion of the ion channel responsible for?

for the detection of changes in transmembrane potential that trigger the opening or closing of the channel

Movement of the voltage-sensor triggers a...

conformational change of the gate of the conducting pathway (controls the flow of ions through channel)

Inactivation of ion channels occurs within ________________ after opening. Inactivation is mediated by an ___________________ that controls the opening of the pore on the inside of the cell

- milliseconds

- intracellular gate

Ligand-gated ion channels

- activated by....

- activation of channels is responsible for the majority of....

- specialized ion channels are activated by....

- activated by binding of ligand to specific site

- activation of channels is responsible for the majority of synaptic transmission by neurons both in the CNS and in the periphery

- specialized ion channels are activated by intracellular small molecules (IP3-sensitive Ca2+ channel, ER) (structurally distinct from convention ligand-gated channels)

Examples of ligands (neurotransmitters) of ligand-gated ion channels (3)

- acetylcholine (Ach) (Nicotinic cholinergic receptor)

- Gamma-AminoButyric Acid (GABAA)

- Glutamate

The activation of the nicotinic ACh receptor allows the passage of both....

NA+ and K+ ions

Synapse

a gap that is present between two neurons

Action potentials are communicated across this synapse by......

synaptic transmission (neurotransmission)

The neurotransmitter diffuses across the synaptic cleft and binds to ___________________***** in the postsynaptic membrane, resulting in ________________ or ____________________ of the postsynaptic neuron

- ligand-gated ion channels****

- depolarization or hyperpolarization

Metabotropic receptors are ____________ linked with ion channels on the membrane of the cell, often through ___________

- indirectly

- G protein coupled (transducer)

G protein-coupled receptors are inherently...

metabotropic

Metabotropic receptors (4)

- what is the most common?

- affects.....

- the 2nd messengers can alter....

- G-protein-coupled receptor (most)

- affects ion channels & 2nd messengers

- 2nd messenger can alter enzyme, effector proteins & DNA expression (effects on cell functioning)

- slow/long acting

Ionotropic receptors (4)

- permits flow of ions

- ligand-gated, (as opposed to voltage-gated channels in action potential)

- neurotransmitter can cause channel to open or close

- fast/short acting

G-Protein Coupled Receptors (GPCRs)

- signal transducers

- convey information (agonist binding) to effector proteins

- (effectors include adenylyl cyclase, phospholipases, plasma ion channel (Ca2+, K+)

The GPCRs are a large family of signaling molecules that are known to...

activate heterotrimeric G proteins to regulate downstream effectors

Effectors for G proteins

adenylyl cyclase, phospholipases, plasma ion channel (Ca2+, K+)

The agonist-induced GPCR activation is a change in what?

the relative orientations of the TM helices leading to a wider intracellular confirmation changes (crucial to signal transduction function)

(important intracellular sites: PKA and GRK phosphorylation sites)

Inverse agonists and antagonists eventual effect in GPCR but be the....

prevention of this TM helix reorientation (conformation change)

The G-Protein Regulatory Cycle (6)

1. Drug binds receptor

2. induces conformational change/activation of receptor

3. G-protein cycle initiated with GTP replacing GDP

4. the heterotrimer dissociates from receptor, and activates effectors/signaling

4. GDP will now replace GTP on the G-protein

5. the heterotrimer re-assmbles and re-couples to the receptor

6. ready to start the signaling cascade again when drug binds

G Protein-coupled Receptors

Upon activation by a ligand, the receptor binds to a partner_______________________ and promotes exchange of _______ for ______, leading to ________________ of the G protein into α and βγ subunits that mediate _________________

- heterotrimeric G protein

- GDP for GTP

- dissociation

- downstream signals

Enzyme/Kinase-Linked Receptors

- linked directly or indirectly to an enzyme (or enzymatic activity)

- signal transduction requires this enzyme/enzyme activity

- slow acting (hours-days)

Protein kinase receptors

high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones

Majority of kinase receptors are....

receptor tyrosine kinases (RTKs).

(a small group of kinase receptors are receptor serine/threonine kinase (RTSK))

A transmembrane receptor (R) whose intrinsic enzymatic activity (in its cytoplasmic domain) is allosterically regulated by a ligand that binds to its _______________________, leading to __________________ and thus ________________

- extracellular domain

- R dimerization and thus R activation

autophosphorylation***

- RTKs are activated by ligand-induced oligomerization (dimerization), which juxtaposes the cytoplasmic tyrosine kinase domains

- The activated tails then phosphorylate each other on several tyrosine residues

Enzyme-Linked Receptors: Interferon Gamma Receptor (IFNY)

- IFNY binds to receptor; leads to dimerization

- JAK is activated and phosphorylates Stat1

- Stat1 forms dimers and becomes active

- active stat enter nucleus and activates transcription

Enzyme-Linked Receptors: Guanylyl Cyclase Receptors

enzyme Guanylyl Cylase (GC) is a receptor, with an extracellular binding domain and intracellular guanylyl cyclase (catalytic/enzymatic) domain

- cGMP is an important second messenger, internalizing messages by intracellular messengers like peptide hormones and NO. can also function as an autocrine signal

Cytosolic guanylyl cyclase form (soluble form) is a receptor for....

nitric oxide (NO)

Intracellular receptors

- located inside cell (or even inside nucleus)

- ligand will have to penetrate the plasma membrane (lipid-soluble) to reach its receptor

- ligand binding induces gene transcription

Examples of intracellular receptors (2)

- nuclear receptors

- IP3 receptor located on the ER

Intracellular receptor ligand examples (2)

- classic hormones (thyroid & steroid hormones)

- intracellular second messengers like inositol triphosphate (IP3)

A ________________________ ligand/drug can cross the membrane to act on an intracellular receptor

lipid-soluble (hydrophobic) (enzyme or regulator of gene transcription)

Nuclear receptor may contain...(3)

- ligand binding domain

- DNA binding domain

- regulatory domain

Nuclear receptors (NRs)

class of transcription factors responsible for sensing steroid and thyroid hormones and other messenger molecules

Activated NRs regulate....

expression of specific genes

(controlling the development, homeostasis, and metabolism)

Nuclear Receptor Type I*****

- located in the cytosol in the absence of ligand

(in the presence of ligand, moves to nucleus)

Example of nuclear receptor type I (5)

- hormone binding to the NR triggers dissociation of heat shock proteins (HSP)

- leads to dimerization and translocation to the nucleus

- in the nucleus, the NR binds to a specific sequence of DNA known as a hormone response element (HRE)

- the NR DNA complex recruits other proteins responsible for transcription of downstream DNA and mRNA

- eventually translated into a protein and results in a change in cell function

Nuclear Receptor Type II****

- located in the nucleus bound to DNA regardless of ligand-binding status

Example of nuclear receptor type II, thyroid hormone receptor (3)

- TR is heterodimerized to the RXR

- in the absence of ligand, the TR is bound to corepressor protein

- ligand binding to TR causes a dissociation of corepressor and recruitment of coactivator protein, which recruits additional proteins such as RNA polymerase responsible for transcription of downstream DNA into RNA and eventually a protein

Thyroid hormone receptor

1. T4/T3 penetrate the membrane

2. T4 is converted to T3, which in turn binds to the thyroid receptor (TR)

3. T3/TR complex translocates to the nucleus

4. the complex binds the response element of the gene and initiates gene transcription

(thyroid gland produces 3 hormones: calcitonin, thyroxine (T4), and triiodothyronine (T3). T3 and T4 have similar function, involved in control of metabolism. calcitonin is involved in regulating the levels of blood calcium.

T3 and T4 are collectively referred to as the thyroid hormones)

Catalyst

accelerates the process but it not consumed by the reaction

Receptors as "Catalysts"

- enzymatic receptor

- ion channel (flow of millions of ions)

- receptors for steroid hormones (NRs-transcription of copies of mRNA)

Receptors and their associated effector/transducer proteins act as ___________________ from multiple ligands

integrators of information

Example of convergence

signals transmitted from a GPCR, integrin receptor, and a RTK all converge on RAS, activating the MAP kinase cascade

Convergences in resistance to targeted therapy: BRAF-mutant melanoma

- drug blocks BRAF, but it can still go through other mechanisms

In BRAF-mutated melanoma, a GPCR-cAMP-CREB signaling axis can substitute for MAPK signaling to sustain an MITF-driven program

- MITF amplification can render MITF independent of MAPK signaling

Due to the variety of pathways regulating the same physiological function, a patient may be treated with......

one or several drugs that alter signaling through these pathways

T/F: There are many mechanisms used to transduce intracellular signal cascades

FALSE

there's a limited number of mechanisms

Crosstalk between signaling pathways:

one or more components of one signal transduction pathway affects another

- (allows for convergence in some cases)

Convergence ****

multiple signaling pathways converging on the same target

What will happen when 2 different receptors have opposite effects in the cell?

They tend to negate one another

Signals transmitted from a GPCR, an _________________, and a ______________ all converge on _________, and then activated the ____________________________

- integrin receptor

- receptor tyrosine kinase

- Ras

- MAP kinase cascade

How were receptors identified? (4)

1. Structure-activity relationship

2. Binding of radioactive ligand

3. Molecular cloning

4. Genome project (reverse pharmacology)

Example of structure-activity relationship: Epinephrine and norepinephrine*****

epinephrine and norepinephrine differ only by one methyl group

- both can stimulate the heart with roughly equal potency

- epinephrine is a much better bronchodilator

Cardiac cells contain predominantly _______________________. whereas bronchial smooth muscle cells contain __________________

- B1-adrenergic receptors (norepinephrine targets)

- B2-adrenergic receptors (epinephrine targets for bronchodilation, this is why it is a better bronchodilator)

Characterization of receptors diagram

Major types of drug receptors chart

Receptors are subject to....

feedback regulatory controls

Desensitization (refractoriness or downregulation) often follows...

continued stimulation with agonists

T/F: Desensitization is only homologous

FALSE

may be homologous or heterologous

What does desensitization mechanism involve? (3)

receptor phosphorylation, relocalization (internalization), alteration in rate of synthesis

Desensitization can result from temporary ________________ of the receptor to _______________ or from fewer ___________ being synthesized

- inaccessibility

- agonist

- receptors

Homologous desensitization

adding an agonist for receptor A, desensitizes receptor A (tolerance)

- leads to phosphorylation of receptor

Heterologous desensitization

adding an agonist for receptor A, desensitizes receptor B (DDI)

- triggers phosphorylation of a different receptor

Receptor initial response, desensitization, recovery chart

- Upon exposure to an agonist, the initial response usually peaks and then decreases to approach some tonic level, elevated but below the maximum

- If the drug is removed for a brief period, the state of desensitization is maintained such that a second addition of agonist also provokes a diminished response

- Removal of the drug for a more extended period allows the cell to "reset" its capacity to respond, and recovery of response usually is competing