Receptors and Signal Transduction

receptors

proteins that bind to ligand that results in cell behavior

short-term and long-term changes after receptor activated

(depends on the purpose of signaling molecule)

short-term: cytoplasm only; change in enzyme activity

long-term: nucleus; increase in protein abundance

-requirement for translocation of molecule into nucleus

receptor-mediated endocytosis

uptake of extracellular materials bound to specific cell-surface receptors by infolding of plasma membrane to form small membrane-bound vesicle

ligand

naturally-occurring or synthetic analog that binds to receptors

Agonist vs. Antagonist ligands

agonist: turns on receptor

antagonist: turns off receptor; blocks function of natural molecule

categorizing ligands

distance traveled, lipid solubility

categories of ligands (distance traveled)

plasma-membrane associated protein: signaling cell binds to adjacent target cell; no movement

synaptic: via synaptic cleft

autocrine: target site of extracellular signal is on same cell

paracrine: secretory vesicle from secretory cell --> signal travels to target cell

endocrine: signal travels from gland to target via bloodstream

pheromonal: signals via secreted pheromones

autocrine cells examples

some cancer cells autocrine in nature: divide by secreting their own growth factors

eukaryotic cell cultures: secretes sufficient growth factors with high density cell culture

Athena Pharmaceuticals

supplement that guarantees a loving, monogamous relationship by intensifying your pheromones

membrane-soluble ligands

receptors soluble within cell membrane

-steroid superfamily

steroids

membrane soluble ligand with receptor in cytoplasm

-cortisol, testosterone, estradiol, vitamin D3

vitamin d3 correlations

sufficient vitamin D levels linked with less severe COVID cases, low rates of non-skin cancers

steroid transport into cell

inhibiting complex prevents activity of receptor --> steroid added --> changed shape ejects inhibitory complex --> receptor with ligand to nucleus --> transcription (primary and secondary)

primary and secondary nuclear responses to steroid attached to protein receptor

primary: shuts off primary-response genes, turns on secondary response genes

secondary response: proteins created

hsp70 and hsp90

inhibitory complex ejected after steroid bind to receptor

membrane-insoluble ligands

cannot diffuse through cell membrane; receptors on outer cell membrane to interact

structures that carry membrane-insoluble ligands into cell

ligand-gated ion channel, receptor-associated endocytosis (RAE), receptor-associated kinases, cytosolic kinases

receptor-associated endocytosis

internalize key molecule(s) to be used by cell or internalize toxic proteins (lysosome dissolution)

receptor-associated kinase

cytokine receptors, receptor tyrosine kinases (RTKs)

-defect in over half of cancers (stimulates secretion of growth factors

cytosolic kinases

G-protein coupled receptors (GPCR; used in many medications)

Nictotinic acetylcholine receptors

ligand-gated ion channel

channel closed --> acethylcholine binds to receptor --> channel opens for ions to fuse in/out

ferrotransferrin

RME: transferrin with iron

transport of iron into cell process

RME

binding of ferrotransferrin to receptor --> clustering (receptor/ligand together in coated pit) --> coated pit falls off, coated vesicle results) --> early endosome --> protons pumped into endosome, intracellular pH increased --> late endosome --> increased acidity causes iron to be released --> ligand and receptor recycled back to cell membrane (dynamin) --> repeat

coated pit formation

ligand binds to receptor --> pit forms, adapter complex surrounds pit --> triskelion clathrin self-assembles around this structure

clathrin

protein that coats the plasma membrane's inward-facing surface and assists in forming coated pits (endocytosis)

-triskelion

triskelion

structure formed by clathrin molecules consisting of three polypeptides radiating from a central vertex; the basic unit of assembly for clathrin coats

dynamin

GTP-dependent kinase required for release of clathrin-coated vesicles from membrane

important of acidification of early endosome in endocytosis

allows for the expulsion of material within the ingested vesicle

experiments testing the importance of increasing endosomal acidity

poison proton pump; culture cells, dilute with NaCl; add drug hydroxychloroquine

-proton-pump inhibited

viruses infection via receptor-mediated endocytosis

infects cell in via RME

-COVID infection of cells via LDL-RME; hydroxychloroquine to prevent virus release from endosome

receptor-mediated endocytosis LDL process

cell-surface LDL receptor binds to apoB protein of LDL particle --> LDL binds --> NPXY sorting signal/AP2 complex prompts cell to undergo RMS --> Clathrin-coated pits invaginate into the cytosol, bringing with it any LDL attached to LDL receptors --> proton pumping to acidify endosome --> LDL receptors to release LDL in an early endosome --> LDL receptors are returned to the plasma membrane --> early endosome, with LDL, matures to a late endosome --> late endosome fuses with lysosomes --> acid hydrolases LDL, releasing cholesterol

difference between ferrotransferrin RME system and LDL RME system

ferrotransferin binds directly to cell receptor

LDL binds to its receptor --> receptor binds to AP2 complex

LDL particle strucure

cholesterol, apolipoprotein B (apoB), phospholipids

apoB

proteins on belt that combines with an LDL receptor found in clathrin-coated pits on the cell membrane

investigation of how LDL particles enter cells

pulse-chase: LDL labeled with radioactive 125-iodine

cultured cells incubated with 125-I-labeled LDL --> track level of particles bound to the surface of cells, internalized, and transported to lysosome to be degrading through time

familial hypercholesterolemia (FH)

inherited condition (autosomal dominant) in which extremely high levels of LDL particles remain in bloodstream

-visible cholesterol deposits, statins not effective

potential causes of FH

mutations in LDL receptor gene: LDL receptor not synthesized at normal rate (deficiency), receptor not transported properly from RER to cell membrane, receptor not recycled properly

defect in apoB (protein belt of LDL particle that binds with receptor)

clustering: defect in NPXY sorting signal (no signal to start RMS)

NPXY

a small part of the LDL receptor containing the NPXY sorting signal which will be important in recruiting AP2 coat proteins

Alzheimer's and LDL receptors in the brain study

high concentrations of LDL receptors in brain found to protect mice from Alzheimer's

-crossed transgenic Alzheimer's mouse with high-LDL brain receptor --> progeny still developed Alzheimer's but less so (less tau protein generated in neurons)

FH studies

mouse with chimeric liver 95% humanized through transfer of LDL-diseased human cells "cured" through single does transfection of human wild-type LDL receptor

G-protein coupled receptors (GPCRs)

a signal receptor protein in the plasma membrane that responds to the binding of a signaling molecule by activating a G protein

orphan receptors

receptors that have no known ligand (GPCRs)

common elements of GPCRs

-serpentine: membrane-imbedded receptor that contains seven membrane-spanning helices

-associated with a heterotrimeric G protein (alpha, beta, gamma subunits)

-functions as receptor-activated switch by cycling between active GTP-bound and inactive GDP-bound states

-participate in amplification and desensitization of signaling pathway

serpentine

GPCRs

snakelike in shape or movement; winding as a snake

cell responses associated with GPCRs

short-term responses by quickly modifying activities of existing proteins

GPCR process

agonist binds to inactive GPCR; GDP-bound G protein (inactive) --> GPCR activated and binds to alpha unit of G protein; dissociation of GDP and binding of GTP in alpha unit --> active G protein dissociates from receptor, diffuses along membrane and binds to effector enzyme --> signal sent throughout cell --> G protein available for reuse; GTPase function of G protein dephosphorylates GTP to inactivate alpha unit, trimeric complex restored --> repeat once new signaling molecule received by GPCR

Robert Lefkowitz and Brian Kobilka

won Noble Prize in chemistry in 2012 for the discovery of G-protein couple receptor family

studying techniques for GPCRs

X-ray crystallography, radioligand binding assay, measuring second messenger effect of GPCR stimulation, nanodisks

X-ray crystallography (GPCRs)

analyzes diffraction patterns coupled to analytical programs to deduce structure

radioligand binding assay

used to study receptor-ligand Interactions

incubate cells with radiolabeled ligand --> wash away unbounded ligands (centrifugation) --> measure the signal left on the cell --> total Binding A --> perform the same experiment but increasingly add separate unlabeled ligand --> measure concentration of labeled ligand that remains after a given concentration

GTPase switch proteins

class of proteins that cycle between active (GTP) and inactive (GDP) states

-GEF: GDP is exchanged for GTP (activate)

-GAP: GTP is hydrolyzed to GDP (deactivate

Guanine nucleotide exchange factors (GEFs)

GTPase switch protein; stimulate the exchange of GDP for GTP thereby promoting G-protein function

GTPase-activating proteins (GAPs)

GTPase switch protein; increasing the rate of GTP hydrolysis of GTP-bound proteins (deactivate)

G-protein cascade and FRET

analyze if there is a dissociation of a trimeric G protein; dictyostelium amoebae cAMPs

Dictyostelium (amoebozoa) use in understanding G-protein cascade

cAMP acts as an extracellular signaling molecule for self-growth that binds to and signals via GPCR

Dictyostelium (amoebozoa) protein cascade and FRET interpretations of results

YFP on GPCR, CFP on receptor

yellow fluorescence: GPCR remains inactive, docked on receptor (wavelength transfer from CFP)

cyan fluorescence: GPCR active; complex dissociated

muscarinic acetylcholine receptors

G-protein-coupled receptors that bind acetylcholine; slows heart rate

-agonist is muscarine

visual transduction

photons --> release of neurotransmitters --> vision

inner and outer segments of rods

inner: synaptic terminal, nucleus, organelles

outer: disks containing photopigment rhodopsin; synthesized at area closest to organelles, climb to end of rod where they are shed

retinal pigment epithelium (RPE)

area in which the photoreceptors meet with the epithelial cells; where disks of rods are shed)

rhodopsin

light-sensitive GPCR in rod cells that causes light sensitivity

components of rhodopsin

opsin and 11-cis retinal

retinal after rhodopsin activation/during inactivity

no signal: bound 11-cis-retinal locks rhodopsin in inactive form

signal: 11-cis-retinal isomer rapidly converted to trans isomer

-shift in conformation noted by RPEs

opsin

the protein portion of visual pigment molecules

uniqueness of rhodopsin receptors relative to other receptors

activation is triggered by absorption of photon light, not by the binding of a ligand

Why is the standard rod cell potential more positive than that of typical neurons?

rod cells in the dark release neurotransmitters constantly to signal to brain to perceive darkness

-membrane of resting rod cell remains in depolarized state because sodium and calcium channels (nonselective cation channels) are open

opening of nonselective cation channels in rod cells

sodium and calcium channels remain open when cell is in depolarized state

-open in response to binding of secondary messenger cyclic guanosine monophosphate (cGMP)

exposure of light to depolarized rod cell

lowers concentration of cGMP (closing of nonselective cation channels) --> membrane potential become more negative (repolarization) --> reduction of neurotransmitters released

rod cells when depolarized/polarized

depolarized: relatively high cGMP concentration, nonselective cation channels open; frequent release of neurotransmitters (constantly signaling darkness)

polarized: relatively lower cGMP concentration, nonselective cation channels close; lower rate of release of neurotransmitters

cGMP phosphodiesterase

an enzyme in cells that converts cGMP into GMP

-occurs in repolarization stage

arrestin

enzyme that participates in desensitization of GPCRs by phosphorylating them after they have been activated by ligand binding

dark adaptation

the recovery of the eye's sensitivity to visual stimuli in darkness after exposure to bright lights

-restores retinal binding site to 11-cis-retinal to inactivate

-rhodopsin kinase, arrestin to rhodopsin

rhodopsin kinase

phosphorylation of rhodopsin when it is in its active

-dark adaptation

arrestin-rhodopsin complex

dramatically speeds up process of dark adaptation

-completely prevents the formation of G protein complex and further activity of PDE

transducin

G-protein that couples rhodopsin to the enzyme phosphodiesterase in rod photoreceptors

transducin and arrestin concentration on outer segment of rod during light and dark adaptation

light: 90% transducin, 10% arrestin

dark: 90% arrestin, 10% transducin

receptor tyrosine kinases

receptor protein spanning the plasma membrane; the cytoplasmic (intracellular) part of can catalyze the transfer of a phosphate group from ATP to a tyrosine on another protein

Erythropoietin (EPO)

cytokine that prompts the division and differentiation of RBCs

-can use Western blots to distinguish phosphorylated from dephosphorylated proteins activated by EPO

RTKs examples

insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF), mitogens

JAK pathway

single ligand binds to adjacent surface tyrosine-associated receptors (JAKs)--> dimerization and phosphorylation of activation loop tyrosines

RTK pathway

two ligands bind to adjacent RTKs --> dimerization activates protein tyrosine kinases --> phosphorylation of additional tyrosine residues (activation loop)

RTK pathway and cancer

5% of cancers have defects in pathway

-defective RAS: constitutive (always active; cell always dividing)

RAS

monomeric G protein; constitutive when defective (cancer)

HER2 receptor

a growth factor receptor located on the surface of epithelial cells in many tissues; made in excess in certain types of breast and ovarian cancers

Herceptin

mAB to treat HER2+

-first FDA-approved biologic used to treat cancer

-advanced to Kadcyla

DM1 (Herceptin)

microtubule disruptor; blocks mitosis by altering mitotic spindle

Insulin (RTK)

increases glucose transport through GLUT4

measuring efficacy of insulin treatment

add 3H-glucose to treatment and control groups, measure tracer over time

autophosphorylation

the process by which one molecule of the tyrosine kinase receptor dimer phosphorylates its partner

proving autophosphorylation is required for insulin signal transduction

mABs, site-directed mutagenesis

autophosphorylation domains

amino acid onto which ATP binds

tyrosine: area on protein that is phosphorylated

mABs to prove autophosphorylation in insulin signaling

mAB that prohibits phosphorylation of insulin receptor --> binds --> decreases signal transduction

site-directed mutagenesis

change a specific DNA code to change a protein

site-directed mutagenesis (insulin signal transduction)

autophosphorylation is required

lysine (AA to which ATP binds) --> aniline (ATP cannot bind) --> decrease signaling

tyrosine (protein that is normally phosphorylated) --> phenylalanine (no phosphorylation) --> decreased insulin signaling

Doug Melton

creation of functional human pancreatic beta cells in vitro to treat Type I diabetes

-founder of Harvard Stem Cell Institute

GEN

pancreatic tissue engineering for diabetes

Erivedge

treatment for metastatic basal cell carcinoma

-takes advantage of Hedgehog

Hedgehog pathway

signaling pathway plays an important role in the regulation of cell differentiation and organ formation during normal vertebrate embryonic development

secondary messengers

relay signal from ligand-receptor to cell

examples of secondary messengers

cAMP, cGMP, IP3/DAG/PKC, Ca2+/Calmodulin, Nitric oxide

-steroid are not SMs (deliver signal itself)