Chapter 6 - Signaling Pathways

Common Components of Signaling Pathways

-Receptor-Ligand Interactions

-GTP binding proteins

-Protein kinases and phosphatases 

Kinases in Signaling Pathways

-Activation of most cell surface receptors leads to changes in protein phosphorylation

-Receptors themselves may possess kinase activity as well

-Ligand-binding to receptors activates kinase 

Fast vs Slow Responses

-Fast pathways contain all proteins necessary, therefore activity/function of proteins change in response to a signal

-Slow pathways require target proteins to be synthesized, therefore expression of proteins changes in response to a signal

-Long term responses (slow pathways) consist of infrequent events such as cell division, differentiation, death, cellular communication, and deevelopment

-Long term responses often involve Tyrosine-Kinase receptors

Tyrosine-Kinase Receptors

-Single transmembrane domain

-Extracellular ligand binding domain

-Intracellular kinase domaind (tyrosine-kinase enzyme with a tyrosine tail)

Activation of Tyrosine-Kinase Receptors

1. Ligand binding results in receptor dimerization, two copies of the receptor will forma dimer

2. Receptor subunits associate, resulting in cross-phosphorylation (both receptors will be phosphorylated) 

3. Phosphorylation receptoors are now activated, receptor recruits proteins and binds through the phosphorylated tyrosine

4. Specific intracellular proteins will recognize and bind to the phosphorylated tyrosines through their SH2 domains

-Pathways that are turned on depend on the type of recruited SH2 domain proteins 

Regulation of Cell Division

Mitogens - Proliferative chemicals that activates the Ras/MAPK pathway

-Growth factors like EGF, PDGF, often activates the Ras/MAPK pathway (Mitogen-Activated Protein Kinase = MAPK)

-Activation of this pathway leads to cell division

Initial Activation of Cell Division

1. Tyrosine-Kinase Receptor is activated

-Ex; EGF ((ligand) binds to the EGFR (EGF receptor)

2. Adaptor proteins are recruited to the receptor

-Ex; GRB2 (via its SH2 domain)

3. Ras-GEF protein is recruited (Guanine Exchange Factor Protein that works on the substrate ‘Ras’ to activate it)

-Ex; Adaptor protein (GRB2) recruits Ras-GEF (called ‘sos’ in the pathway)

4. When activated by binding to GRB2, SOS exchanges GDP for GTP on Ras

-Ras (small G-protein) is now active, resulting in the activation of a kinase cascade

Activation of Cell Division Kinase Cascade

-Ras results in the phosphorylation of MAPKKK (active now) which phosphorylates MAPKK which in turn phosphorylates MAPK

-Once MAPK is phosphorylated, it will add phosphates to protein targets/factors

Common MAPK Targets

-MAPK often targets transcription factors that are activated by phosphorylation

-These transcription factors turn on specific genes, resulting in protein expression

-Example; Transcription factors trigger the synthesis of proteins involved in cell division

• Polymerase, Histones, Helicase, Topoisomerase 

G-Protein Couples Receptors

-Very common and abundant receptor used in fast-acting pathways

-Human genome has 900 types of GPCRs used in various fast responses (sensory responses, neurotransmitter response, hormone receptors, macromolecule metabolism) 

-Often related to short-term changes in cell function

GPCR Structure

-Contains seven transmembrane domains

• N-Terminus; Extracellular domain with NH3+

• C-Terminus; Intracellular domain with COO-

-Once activated, the G-protein attaches at the cytoplasmic loop between the 5th and 6th transmembrane domains (C3)

Heterotrimeric G-protein

Contains 3 different subunits; alpha, beta, gamma

-G-protein activation changes activity of membrane bound effector protein and often leads to creation of second messengers

-Complex associates with the membrane in GDP bound ‘off’ conformation (peripheral membrane)

Activation of GPCRs

1. Ligand binds to GPCR

2. Heterotrimeric G-protein is recruited to the intracellular side of the receptor and binds

3. GDP is then switched for GTP on the alpha subunit

4. G-protein is now activated; subunits dissociate from each other (G-alpha bound to GTP, G-beta/gamma remain together)

• Each complex binds to and/or actives/inhibits targets

5. Activity persists until GTP is hydrolyzed by intrinsic GTPase activity. Reassociation of alpha with beta/gamma will turn off the G-protein

Diversity of G-Proteins

-More than 20 different kinds of ‘large’ G-proteins

-Common downstream targets include; regulation of ion channels and regulation of enzymes in signaling pathways

Second-Messenger Molecules

G-proteins activate effector molecules that are commonly enzymes

-Most common enzyme targets; Adenylyl Cyclase and Phospholicase C

-These enzymes generate second-messenger molecules; intracellular molecules that are rapidly produced during signaling and diffuse to act on intracellular signaling proteins

-Molecules have a low molecular weight, concentration changes (up or down) in response to receptor activation

-They can also bind/modify other pathway proteins

-Highly amplified; one receptor can result in the creation of thousands of second messenger molecules

-Diffuse through the cytoplasm faster than proteins (due to smaller size/low molecular weight)

-Ex; Adenylyl Cyclase produces cyclic AMP (cAMP)