2nd Messenger Systems, continued

Cyclic AMP production and degradation• In resting cells, the cAMP level is so low (10-8M) that it does

not bind the targets, such as regulatory subunits of cAMP-gated channels. Stimulation of a G Protein Receptor raises the level 100x, enough to saturate the receptors.

What if a G-Protein-adenylcyclase system got stuck “on”?

• The cholera toxin is released in the gut by the bacteria Vibrio cholerae. The toxin enzymatically alters Gαs so that it no longer hydrolyzes GTP. The continuous presence of stimulatory Gαs causes the intestinal cells to secrete large amounts of salt and water, causing diarrhea and therefore dehydration.

• The Bordetella pertussus toxin acts in a similar way on the inhibitory G protein, but it is not apparent why it causes whooping cough.

Cyclic AMP modulation of Protein Kinases

Effects of Protein PhosphorylationAddition of a phosphate by protein kinase or removal

by protein phosphatase is the most common post-translational modification of proteins. It turns processes on and off, e.g.,

1. Cell motility2. Membrane channels3. Cell division>99% of phosphorylation occurs on serine or threonine

residues.The effects on structure include:1. Steric interference: altering affinity2. Conformational change that blocks or activates

enzymes3. Creation of binding sites

Regulation of protein kinase A

• The inactive form consists of two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunits induces a conformational change that allows the enzymatically active regulatory subunits to dissociate.

Receptor Tyrosine Kinases

Receptors that are catalytic

The human genome encodes 59 receptors of this type – most are for growth factors. The name receptor tyrosine kinase refers to the fact that the intracellular domain of these proteins has intrinsic kinase activity.

These receptors are unique in that the receptor is a dimer. In some cases, interaction of the 1st message with the extracellular domains of two receptor molecules causes formation of the dimer. The intracellular domains then phosphorylate each other. This activates the receptor, which can proceed to phosphorylate particular tyrosines of other proteins. In other cases (the insulin receptor), the receptor is already a dimer and insulin binding simply induces a conformational change.

Structures of receptor protein tyrosine kinases (FYI: PDGF is platelet derived growth factor, EGF is epidermal growth factor)

Response sequence• 1. Ligand-induced receptor dimerization

• 2. Autophosphorylation: polypeptide strands cross-

phosphorylate one another.

Response sequence, con’t.• 3. This increases protein kinase activity AND• 4. Phosphorylation of tyrosine residues creates binding sites for additional

proteins that transmit signals downstream. (SH2 is the region that binds)• 5. The activation of the downstream signaling molecule is the first step in the

growth factor responses.

Link between a Receptor Protein Tyrosine Kinase and a second-messenger system: Phospholipase C

• The SH2 domain of Phospholipase C-γ allows it to associate with PTK and be localized near the membrane, where it can attack a specific kind of membrane lipid, turning it into 2 signaling molecules.

• (The same reaction is generated by G-Protein activation of Phospholipase C-β.)

• The target of Phospholipase C, Phosphotidylinositol 4, 5 bis phosphate, is a minor membrane component, mainly found on the inner half of the bilayer. Phospholipase C catalyzes phosphotidylinositol 4,5-bisphosphate (PIP2) conversion to the second messengers inositol trisphosphate (IP3) and the corrresponding diacylglycerol (DAG). This reaction requires phosphate donation by ATP.

Inositol Triphosphate (IP3) and Diacylglycerol (DAG) as second messengers

Ligand-triggered sequence

One effect of IP3: release of Ca++ from the endoplasmic reticulum

Control of Cell Function by 2nd Messenger Systems

An example: Glycogen metabolism

A push-pull hormonal system regulates plasma glucose levels

• Glucagon and epinephrine are released when plasma glucose levels fall below about 5 mM

• Insulin is released when plasma glucose levels rise above about 5 mM, and in response to gut signals that indicate ingestion of a carbohydrate meal

2 tissues, 3 hormones

tissue Hormones Receptors/2nd messengers

Net effects on glycogen

Liver Glucagon (from pancreatic alpha cells)

Glucagon receptor/Gs G protein/cAMP

degradation

Epinephrine (from adrenal medulla)

β/ receptor/Gs G protein/ cAMP

degradation

α1 Gq G protein/ IP3 /Ca++

degradation

Muscle Insulin (from pancreatic beta cells)

Insulin receptor/IRS proteins

synthesis

Epinephrine β receptor/Gs G protein/ cAMP

degradation

Insulin has multiple intracellular consequences

• The insulin receptor phosphorylates a family of IRS (insulin receptor substrate) proteins, which then activate other downstream signaling proteins, leading to a large variety of metabolic effects in the target cells.

Major insulin effects that relate to glycogen metabolism in liver

• Enhances activity of glycolytic enzymes (hexokinase, phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase)

• Inhibits glucose-6-phosphatase• Stimulates conversion of glycogen synthetase kinase

from active to inactive form• Since glycogen synthetase kinase inactivates glycogen

synthetase, inactivating it stimulates glycogen synthetase

• Inhibits glycogen phosphorylase• (glucose uptake by liver is mainly via the insulin-

insensitive GLUT2 transporter)

Effect of insulin on liver

Glycogen synthesis and glycolysis are stimulated; gluconeogenesis is inhibited

Major effects of insulin on glycogen metabolism in muscle

Just as in liver, except that

• gluconeogenesis does not occur in muscle

• in muscle insulin stimulates insertion of insulin-sensitive GLUT4 glucose transporters into the plasma membrane

Effect of insulin on muscle

Glucose uptake, glycolysis and glycogen synthesis are stimulated – gluconeogenesis is not an issue in muscle

Other metabolic effects of insulin

• Stimulates translation of mRNA into protein

• Inhibits proteolysis

• Stimulates enzymes involved in triglyceride synthesis and inhibits lipolysis

• Stimulates expression of genes involved in tissue growth

Major effects of glucagon on glycogen metabolism in liver

Glucagon antagonizes the effects of insulin

Promotes net glycogen breakdown: inhibits hexokinase and glycogen synthetase and activates glycogen phosphorylase and glucose-6-phosphatase

Promotes gluconeogenesis by stimulating key enzymes in the pathway – particularly ones related to fructose phosphates, as shown in following slides:

Glycogen synthetase a

Protein Kinase AcAMP

Phosphorylase kinase b Phosphorylase kinase a

Glycogen synthetase b

Glycogen synthesis activity decreases

Glycogen breakdown activity increases

Glucagon and epi stimulate glycogen mobilization by activating cAMP-dependent protein kinase A

Active forms in black; inactive forms in red