BME 429 - Receptor and Ligand Interactions

North Carolina State University - BME 429 (Cellular Engineering) - Dr. Ashley Brown

Cell

The basic biological unit of living organisms. Tissue and development changes occur at the cellular level

What do membrane-bound organelles do for animal cells?

Enhance compartmentalization and regulation of cell functions by separating various biochemical processes.

What are some reasons why we might want to control cell signaling?

• Stem cell differentiation

• Immune engineering

• Targeted cell therapies (ie, cancer)

• Preventing/treating fibrosis

• Cell manufacturing

Paracrine signaling

Cells communicate over relatively short distancesthrough the release of signaling molecules that affect nearby target cells.

Synaptic signaling

A type of paracrine signaling, where nerve cells transmit signals over a synapse.

Autocrine signaling

A cell signals to itself.

Endocrine Signaling

Long-distance cell signaling. Signals are produced by specialized cells and released into the bloodstream, which carries them to target cells in distant parts of the body.

Cell-Cell Contact

Cell signaling through gap junctions or cell membrane protein interactions.

Ligand

• A molecule used for cell signalingthat binds to a receptor on a target cell, initiating a response.

• Binds to just 1 or a few target receptors

• Are often proteins, but can also be ions and phospholipids

Receptor

• A receiving molecule/protein on the target cell that binds to a specific ligand, triggering a cellular response.

• Recognizes just 1 or a few specific ligands

How does a ligand bind to receptor?

• Binding of a ligand to a receptor changes its shape or activity, allowing it to transmit a signal / directly produce a change inside the cell

• Doesn’t use covalent bonds nor chemical reactions

Upstream

Used to describe molecules and events that come earlier in the relay chain

Downstream

Used to describe molecules that come later relative to a particular molecule of interest in the relay chain

Phosphorylation

• Addition of a phosphate group to one or more sites on the protein

• Phosphate groups are typically linked to one of the 3 AAs with hydroxyl (-OH) groups in side chains (tyrosine, threonine, serine)

• Not permanent

Kinase

An enzyme that catalyzes the transfer of the phosphate group; usually represented by a “K”

Phosphatase

Enzymes that remove a phosphate group from their targets

Intracellular receptors

• Receptors found inside the cell, such as in the cytoplasm or nucleus

• Most of their ligands are small and hydrophobic (must be able to cross plasma membrane to reach these receptors)

• Are unique because they can cause changes in transcription of genes very directly by binding to DNA and altering transcription themselves

Cell surface receptors

• Membrane-anchored receptors found inside the plasma membrane

• Many different kinds of molecules can act as their ligands, since the ligand doesn’t need to pass through plasma membrane

3 domains/protein regions of a cell-surface receptor

1. Extracellular ligand-binding domain

2. Hydrophobic domain extending through membrane

3. Intracellular domain, which often transmits a signal

3 common types of cell-surface receptors

• Ligand-gated ion channels

• G protein coupled receptors

• Enzyme-linked receptors

  • Receptor tyrosine kinases (subclass)

Ligand-gated ion channels

• Ion channels that can open in response to the binding of a ligand

• Contains a membrane-spanning region with a hydrophilic channel through middle

• Allows ions to cross membrane without having to touch hydrophobic core of phospholipid bilayer

• Binding of ligand causes protein structure changes to allow for ion passage or channel closure

• Common in neurons (neurotransmitters as ligands)

G protein-coupled receptors (GPCRs)

• Large family of cell surface receptors that share a common structure and method of signaling

  • 7 transmembrane domains

  • Signals through G protein

• All types of G proteins bind the nucleotide guanosine triphosphate (GTP), which they can break down (hydrolyze) to form GDP

  • G protein active when attached to GTP

  • G protein inactive when attached to GDP

Enzyme-linked receptors

• Cell surface receptors with intracellular domains that are associated with an enzyme

• In some cases, the intracellular domain of receptor is an enzyme that can catalyze a reaction

  • Other enzyme-linked receptors have an intracellular domain that interacts with an enzyme

Receptor tyrosine kinases (RTKs)

• An important class of enzyme-linked receptors found in humans and many other species

• Transfers phosphate groups specifically to AA tyrosine

• In many cases, the phosphorylated receptors serve as docking platform for other proteins that contain special types of binding domains

• Used in growth factor binding

• Activity must be kept in balance: overactive RTKs are associated with some types of cancer

Intracellular ligands

• Steroid hormones (estradiol [female sex hormone], testosterone [male sex hormone], vitamin D)

• Nitric oxide

Extracellular ligands

• Water-soluble: polar or charged

• Mostly peptide (protein) ligands

  • Growth factors

  • Hormones (insulin)

  • Neurotransmitters

Agonist

A drug that causes the receptor to respond in the same way as the naturally occurring substance

Antagonist

A drug that binds to the receptor, but doesn’t produce a response and prevents the receptor binding to the ligand, having an inhibitory effect on the naturally occurring substance

R + L <=> C

Receptor + Ligand <=> Receptor/Ligand Complex

Molecular recognition

• Molecules “recognize” each other when they come close enough to “feel” the presence of each other, either through physically bumping into one another or through interactions of the fields (electrostatic) between each other

• Uses non-covalent interactions, so can be reversible

Specificity

A description of how favorable the binding of the ligand/receptor for the receptor/ligand is compared with its possible binding to other types of receptors/ligands that may also be present

Affinity

• Refers to how strong the binding is (as judged by K_association or K_{disassociation} or ∆G°).

• Association or dissociation constants (K_D) typically referred to as the “affinity” or “binding” constants

k_a

Association rate constant for formation of RL or C, “on rate”, k_forward

k_d

Dissociation rate constant for complex RL or C, “off rate”, k_reverse

Stoichiometry

Refers to how many molecules of ligand can bind to a single receptor

Cooperativity

• AKA synergism, refers to situations where binding of 1 or more molecules to the receptor enhances/weakens the binding of additional molecules to the same receptor

• Cooperative binding effects (both positive and negative) are also known as allosteric effects (eg, cofactors, calcium)

Reversible vs. Irreversible binding

• All non-covalent binding processes are reversible, meaning ligand can both bind to and dissociate from the receptor

• Equilibrium reached when the time following mixing is long compared to the t_1/2 (half-life) binding and dissociation

• However, sometimes non-covalent binding is so tight that the association is effectively irreversible and doesn’t reach equilibrium within the relevant time frame

Kinetics

• Term used to describe both the rates at which processes occur and the field associated with the study of rates

• Binding and dissociation processes will be characterized not only by the equilibrium constants but also by how fast association/dissociation occurs

1:1 Stoichiometry for Receptor/Ligand

The simplest case in which 1 ligand binds to 1 receptor

What are binding and other equilibrium constants related to?

Rates of interchange between states involved in the equilibrium process

Rate

A description of how frequently something happens

Zero order reaction rate (unimolecular rate)

• Independent of concentration

• Per time (e.g. sec^-1 (Hz), min^-1)

• k = [conc/time] (M/s)

• Eg, decomposition of ammonia (happens at high temperatures and is independent of concentration because it has to do more with adsorption to the surface area of platinum

First order reaction rate

• Dependent upon the concentration of a single species

• Δ[concentration]/time (e.g. mM product produced/min) or Δquantity/Δtime

  (e.g. micromoles produce produces/sec)

• k = time^-1 (sec^-1)

• Ex:

  • Production of an enzyme product complex from an enzyme-substrate complex

  • Dissociation of a 1:1 receptor-ligand complex to form free ligand and free receptor (while 2 separate species are proceeded, the rate at which they are produced will be dependent upon a single concentration: that of the complex)

Second order reaction rate

• Dependent upon the concentration of 2 species

• Δ[concentration]/time for either products; rate constant depends on both

  inputs

• k = concentration^-1 * time^-1. For example (M^-1*sec^-1)

• Ex: Binding of a ligand to a receptor to form a 1:1 ligand complex (the rate is dependent on both concentrations; L and R can associate only if they bump into each other, and the probability that they will is determined by their concentrations)

K_a

• Equilibrium association rate = [RL] / [R][L] ← these are concentrations at equilibrium (product over reactant)

• K_a = 1/K_d

K_d

• Equilibrium dissociation rate = [R][L] / [RL] ← these are concentrations at equilibrium

  • (also ‘product over reactant’, but the ‘product’ this time is the unbound ligand/receptor)

• Also = free ligand concentration where the total population of free and complexed receptors will be equal (half maximum binding)

Which is the weakest binder?

Light blue

Formula for influence of temperature on equilibrium

ΔG° = RTln(K_d) = -RTln(K_a)