Publisher Summary This chapter describes the cloning of RGS box-containing cDNAs using degenerate PCR and characterization of the PDZ domain of one of these RGS box-containing proteins, RGS 12, using surface plasmon resonance biosensors and yeast two-hybrid techniques. Novel RGS proteins can be cloned using this degenerate PCR strategy (RGSI2 and RGSI4). These novel RGS proteins, with their multidomain structures, suggest higher-order functionality (beyond Ga-directed GAP activity) in the coordination of signal transduction pathways transacted by heterotrimeric G proteins, monomeric G proteins, and/or tyrosine kinases. The methods herein described for assessing PDZ domain binding specificity should prove useful in the analysis of other PDZ domain proteins including PDZ-RhoGEF, the second RGS protein found to contain an N-terminal PDZ domain. Heterotrimeric G proteins, composed of Gα, Gβ, and Gγ subunits, translate cell-surface receptor/ligand interactions into intracellular signaling cascades via receptor-catalyzed exchange of GDP for GTP on the Gα subunit.