Interaction of cellular proteins with subdomains of the α_2a/d-adrenergic receptor

Signal initiation by G-protein coupled receptors involves receptor, G-protein and effector, However, other proteins in the receptor's microenvironment may regulate the efficiency/specificity of the signalling process via an interaction with intracellular regions of the receptor. To address this possibility, we generated fragments from the 13 loop of oc2A/D-adrenergic receptor (AR) as fusion proteins with glutathione-S-transferase (GST) using pGEX-2T or pGSTag for generation of 32P-Iabeled peptides. The receptor subdomain probes were used to screen for interacting proteins in cytosolic cell extracts (bovine brain) by gel overlay and affinity matrix adsorption. Brain cytosol was fractionated by ammonium sulfate precipitation/ion exchange chromatography and proteins were electrophoresed and transfered to nitrocellulose for incubation with 32P-labeled receptor peptide (K318-G364). Selected elutions indicated potential interacting proteins of Mr {kd) -84, 56, 53, 32 and 3Ü: these proteins were not detected with labeled peptides derived from vector alone. The cytosolic fractions were also evaluated in a solution phase assay using glutathione affinity matrices substituted with receptor subdomains generated from the CX2A/D-AR or the Mj-muscarinic receptor. This approach identified potential interacting proteins of Mr (kd) -105, 56, 48, 34 and 32 that were specific for the α_2A/D-AR subdomain. Such strategies may allow the identification of receptor-associated regulatory proteins important for signalling efficiency/specificity.