Cell Signaling (BIO-203) Lecture 5

Signal amplification occurs in many signaling pathways Receptors are low abundance proteins The binding of few signaling molecules to available receptors require production of tens of thousands or even millions of second messenger or activated enzyme molecules per cell. Substantial signal amplification must occur in order for a hormone signal to induce a significant cellular response. For example, a single epinephrine-GPCR complex causes conversion of up to 100 inactive G αs molecules to the active form before epinephrine dissociate from the receptor.

Binding of a single epinephrine molecule to the receptor induces synthesis of a large number of cAMP molecules, first level of amplification. 2 molecules of cAMP activate 1 molecule of protein kinase A. Each activated PKA phosphorylates and activates multiple product molecules, second level of activation.

Phospholipase C activation GPCRs activates protein kinase C (PKC) a ubiquitous family of serine/threonine protein kinases. The pathway leading to PKC activation starts with a class of GPCRs that interact with and activate Gq G-proteins when the receptor has agonist ligand bound. Activated Gq with GTP bound activates its downstream target phospholipase C (PLC) to hydrolyze the membrane lipid PIP2, producing IP3 and diacylglycerol (DAG). IP3 is water-soluble and diffuses through the cytoplasm to the ER, where it binds to and opens a calcium channel, releasing calcium stores from inside the ER into the cytoplasm. Calcium alters many cellular processes, in part by binding to regulatory proteins such as calmodulin and calcineurin. The interaction of both DAG and calcium with PKC activates its kinase activity and the phosphorylation of many different protein targets alters their activity. The involvement of PKC in cellular proliferation and the cell cycle is indicated by the activity of tumor promoters like phorbol esters as PKC activators.

Iron uptake in different cells Diferric Tf in serum Macrophage Hepatocyte Duodenal enterocyte Endosome pH < 6 Ferritin Ferritin DcytbHCP1 HO Haem Apo Tf in serum Haem from RBCs Ferritin HO Diferric Tf Fe 3+ Fe 2+ Haem FPN1 Fe-Tf-TFR1 MCF hephaestin or ceruloplasmin DMT1 STEAP 3

GLUT Glucose transporter found in the plasma membrane of erythrocytes. 12 GLUTS are encoded by human genomes. Glucose conc. is high in the extracellular medium than in cell. GLUT alternates between 2 confirmational states.

Insulin and glucagon work together to maintain a stable blood glucose level The entry of glucose into pancreatic β cells is mediated by GLUT2 Basal blood glucose level is approximately 5mM. The Km value (an indicator of the affinity of the transporter protein for glucose molecules; a low Km value suggests a high affinity).Km The Km for glucose of GLUT2 is 20mM, a rise in extracellular glucose from 5 mM causes an increase in glucose intake.

The conversion of glucose into pyruvate is accelerated resulted in an increased production of ATP in the cytosol. The binding of ATP to ATP-sensitive K channels closes these channels. Closed channels reduced the efflux of K ions from the cell which triggers the opening of Ca channels. The influx of Ca ions raises the cytosolic Ca levels which triggers the fusion of insulin- containing secretory vesicles with the plasma membrane and secretion of insulin.

How do we measure the affinity of a receptor to its ligand? How a maximal cellular response to a signaling molecule achieved? What do binding assays tell us?