Signaling and desensitization of G protein-coupled receptor are intimately related,. and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta(2)-adrenergic receptor (beta(2)AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharon cerevisiae LM102 cells, and dopamine D-3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta(2)AR, which mediates stimulation of adenylyl cyclase, S49 lyinphorna cys(-) cells that lack the alpha subunit of Gs were used. Stimulation of PAR in these cells failed to increase intracellular cAMP, but P-arrestin translocation still occurred.. suggesting that feedback from PAR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Go alpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced P-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of G alpha o and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes. (c) 2005 Elsevier Inc. All rights reserved.