Recently, evidence has emerged that heptaspanning-membrane or G protein-coupled receptors (GPCR) may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, alpha-actinin, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of adenosine A(2A) receptor (A(2A)R). Colocalization, coimmunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. The A(2A)R activation by agonist induces the internalization of the receptor by a process that involves a rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by Cytochalasin D prevented its agonist induced internalization. The A(2A)deltaCTR, a mutated version of A(2A)R, which lacks the C-terminal domain and did not interact with alpha-actinin, was not able to internalize when activated by an agonist. Interestingly A(2A)deltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A(2A)R trafficking.