Analysis of Receptor Phosphorylation

Abstract

Publisher Summary This chapter describes the various procedures to analyze nuclear receptor phosphorylation. It focuses on the methods used for identifying phosphorylation sites, many of which are directly applicable to the analysis of other post translational modifications, such as acetylation and methylation of receptors, and other types of proteins. The nuclear receptor superfamily is a diverse group of ligand-dependent transcription factors and orphan receptors, whose activities are regulated by reversible phosphorylation. Phosphorylation occurs predominantly on serine and threonine residues in the N-terminal and DNA-binding regions of receptors. The study of nuclear receptor phosphorylation necessitates an understanding of cellular signaling pathways and the way kinases and phosphatases in these pathways communicate with the nuclear receptor signaling pathways to modulate receptor function. Studies of the effects of hormones, drug treatments, cell cycle, and other conditions on receptor phosphorylation often provide new insights into receptor function. There are several types of mass spectrometers, and the techniques utilized to detect phosphorylation sites will depend upon the equipment available. Mass spectrometers measure the mass–charge ratio (m/z) of a molecule. Peptides must be volatilized to be detected by the machine. Receptor deletion and point mutations can be used to identify phosphorylation sites indirectly.