Tracking GPCR biosynthesis and degradation using a nonradioactive pulse chase methodology.

The β2-adrenergic receptor (β2AR) is a prototypical member of the G protein-coupled receptor (GPCR) superfamily of proteins and is one of the best-characterized GPCRs due to its role in several important physiological systems. Because of limited availability of high quality antibodies against GPCRs, much of the work done on β2AR took advantage of heterologous expression systems. Overexpressed proteins may overwhelm the cellular regulatory machinery leading potentially to responses distinct from the native protein. To address this issue we generated a stable cell line with a tetracycline-inducible β2AR tagged with a FLAG epitope, such that we are able to control the quantity of receptor produced. This allows us to induce a discrete pulse of FLAG-β2AR transcription and translation allowing us to follow the complete life cycle of the protein from synthesis as an immature protein to degradation. We show that such limited pulses of receptor expression lead to signaling phenotypes that more closely reflect endogenous signaling events.