Characterization of binding kinetics of AR to Gα protein by surface plasmon resonance.

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A receptor (AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the AR C-terminus from 412 to 316 amino acids (AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length AR levels. As cAMP production is downstream of the first activation event-coupling of G protein to its receptor-investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα proteins, we characterize the association of AR and AΔ316R to Gα with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα affinity for AR was greatest for apo-Gα, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the AR C-terminus (AΔ316R) decreased the affinity of the unliganded receptor for Gα by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.