Bio 225 - topic 6

Unicellular organisms existed about ___ years before the first multicellular organisms appeared

2.5 billion, unicellular organisms can participate in cell signalling

Human genome encodes an estimated ____ receptors

1500

Signal molecules typically exist in low concentrations within an organism, but ….

receptors have a very strong affinity for them

The growth and survival of a cell in a multicellular organism depends on: (cell signalling is involved in all of these processes)

Intercellular communication, monitoring of the environment, the formation of appropriate responses to stimuli

Basic principle of cell signaling

The only cells that can respond to a signalling molecule are those that possess a receptor for that signalling molecule

Paracrine signalling

Signaling between neighbouring cells, local signaling, produced in low amounts

Autocrine signaling

Cells produce signal, then respond to it

Juxtacrine signaling

Contact dependent, signal and receptor are both membrane bound, cells are next to each other

Endocrine signaling (hormones)

Small, hydrophobic signals, produced in relatively large amounts through entire body

Neuronal signaling

Signaling molecules are neurotransmitters, act at a short distance (synaptic cleft), can also be considered long distance due to length of some neurons

Contact-dependent signaling

Contact between a cell surface signalling molecule and a receptor at the surface of the target cell, two cells are in direct contact with each other

Direct signaling

Involves flow of molecular traffic between neighbouring cells, cells connected by gap junctions or plasmodesmata

Cell-surface receptors

Receptor protein is transmembrane or is associated with one

second messangers

Binding signal does not enter the cell, signal is transmitted from the receptor to a small molecule inside the cell. This acts as an intracellular signaling molecule (eg: cAMP, Ca2+ cGMP), feedback can be positive (speed up pathway) or negative (slow down pathway), the pathways activated integrate to create an output

Intracellular receptors

Carrier protein and small hydrophobic signal molecule, intracellular receptor protein in nucleus

Common cell surface receptors

Ion channel coupled receptors, G-protein coupled receptors (most common), enzyme coupled receptors

Receptor sequestration

Signal and receptor are brought into the cell in an endosome

Receptor downregulation

Signal and receptor are brought into the cell in an endosome, signal and receptor are then destroyed by a lysosome

Receptor inactivation

Inhibitory protein binds to receptor to stop it in enzymatic reaction

Signaling protein inactivation

Intracellular signal protein binds to receptor, attracting inhibitory protein to it

Signaling by GTP-binding

OFF - GDP, ON - GTP, GTP binding - mediated by GEF, GTP hydrolysis - mediated by GAP

Signaling by phosphorylation

Kinase - carries out a phosphorylation, phosphorylated = ON, not phosphorylated = OFF, phosphate is transferred from ATP to protein (ON), this releases NADP, two points of regulation: turn off kinase or turn off phosphatase, protein phosphatase - mediates protein hydrolysis (losing P), phosphorylation causes a change in structure and therefore a change in function for the protein

ligand

signal molecule (noncovalent bonds between receptor and ligand)

what is Kd, and what are the ideal values for Kd and Ka

Kd is the dissociation constant, ideal is small Kd and large Ka

exogenous ligand

natural or normal ligand

endogenous ligand

from outside the system

agonist

binds and activates

antagonist

binds and inhibits

At equilibrium, receptor and ligand are …

combined to form receptor ligand complex. Concentrations of each side stay the same

Listeria example

Bacteria found in raw milk products, can grow at refrigeration temperatures, hard to trace source of outbreak, affects two signaling pathways, causes cytoskeleton to grow through the membrane, allowing bacteria to enter cell and escape immune system

G-protein coupled receptors (GPCRs)

Robert Lefkowitz and Brain Kobilka - nobel prize in chemistry (2012) for work with GPCRs. There are 7 alpha helical transmembrane spanning regions in the GPCR. More than 800 GPCRs in the human genome. Estimated that 40-50% of all drugs target GPCR activity

G proteins

Alfred Gilman and Martin Rodbell - nobel prize in physiology or medicine (1994) for work with G-proteins, has 3 subunits

Bacteriorhodopsin

Jacques Dubochet, Joachim Frank, Richard Henderson - nobel prize in chemistry, 2017, phototrophy, moves protons across membrane

Heterotrimeric G-proteins

When alpha subunit is bound to GDP it associates with beta subunit and is inactive, contains an alpha, beta and gamma subunit, alpha and gamma subunits have lipid tails - are bound in membrane, alpha subunit has a GDP-binding site

Mechanism for GPCR activation

Resting state: receptor is not bound to ligand; alpha subunit is bound to GDP and associated with beta subunit. Ligand binds receptor; the receptor binds to G protein; alpha subunits releases GDP and acquires GTP, Alpha and beta subunits separate. Exchange of GDP for GTP causes the alpha subunit to dissociate from the beta subunit. G protein subunits activate or inhibit target proteins, initiating signal transduction events. Both the alpha and beta subunit can potentially act as signalling molecules. More commonly the alpha subunit. The alpha subunit hydrolyzes its bound GTP to GDP, becoming inactive. Alpha subunit can hydrolyze its GTP to GDP. The rate of hydrolysis can be moderated by other proteins. RGS - regulators of G protein signalling - these act as GAPs (GTPase activating proteins). Down Regulation - phosphorylation + binding of inhibitor. Subunits recombine to form an inactive G protein. GPCR acts as a GEF when stimulated by its ligand - exchange GDP for GTP on alpha subunit. G proteins are combinatorial - different combos of alpha, beta and gamma will activate/deactivate different signal pathways

CRE/CREB

PKA regulates gene expression (long term), is an example of allosteric regulation

Cholera

V. cholerae adhere to intestinal cells, and secrete choleragen, and AB exotoxin, the A subunit enter intestinal epithelial cells and activates adenylate cyclase via ADP-ribosylation, this results in hyper-secretion of water and chloride ions from the cells, treatment with oral rehydration, sometimes antibiotics, 50% mortality if untreated

Calmodulin

Can be up to about 1% of a cell's total protein mass

Involved in Ca2+ signalling, highly conserved single polypeptide with four high affinity Ca2+ sites, a 10-fold increase in Ca2+ leads to a 50-fold activation of calmodulin, calmodulin does not have its own enzymatic activity, but rather it binds to and activates other proteins, changes structure upon calcium binding, when bound to Ca2+, able to regulate a number of different protein targets in a cell

Tyrosine kinase receptors

Growth pathways - associated with cancers, most common in multicellular organisms

serine/threonine receptor kinases

Important for development, can occur in cancer

Structure of receptor tyrosine kinases

Unbound, no P-Tyrosines, receptor binding induces multimerization, trans-autophosphorylation activates the receptor

Ras activation

From Rat sarcoma, monomeric GTPase, Sos (son of Sevenless) is the GEF for Ras

MAP kinase

MAP = mitogen activated protein, MAP - transcription factors that regulate gene expression and growth signals, Mammalian cells have 12 MAPK, 7 MAPKK and 7 MAPKKK genes