Probing phosphorylation events in biological membranes: The transducer function

Abstract

The extracellular environment is sensed by receptors in the plasma membrane. Some of these receptors initiate cytoplasmic signaling cascades involving phosphorylation: the addition of a phosphate group to a specific amino acid, such as tyrosine, in a protein. Receptor Tyrosine Kinases (RTKs) are one large class of membrane receptors that can directly initiate signaling cascades through their intracellular kinase domains, which both catalyze tyrosine phosphorylation and get phosphorylated. In the first step of signaling, the ligands stabilize phosphorylation-competent RTK dimers and oligomers, which leads to the phosphorylation of specific tyrosine residues in the activation loop of the kinases. Here we discuss quantitative measurements of tyrosine phosphorylation efficiencies for RTKs, described by the “transducer function”. The transducer function links the phosphorylation (the response) and the binding of the activating ligand to the receptor (the stimulus). We overview a methodology that allows such measurements in direct response to ligand binding. We discuss experiments which demonstrate that EGF is a partial agonist, and that two tyrosines in the intracellular domain of EGFR, Y1068 and Y1173, are differentially phosphorylated in the EGF-bound EGFR dimers.