Characterization of binding kinetics of A 2A R to Gα s 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 (A R) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A R C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A R C-terminus from 412 to 316 amino acids (A Δ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A R 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 A R and A Δ316R to Gα with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα affinity for A R was greatest for apo-Gα , moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A R C-terminus (A Δ316R) decreased the affinity of the unliganded receptor for Gα by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.