Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK₂R) called CCK_2i4svR that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK_2i4svR desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK₂R and CCK_2i4svR and induces intracellular Ca²⁺ ([Ca²⁺]_i) increases. Agonist-induced increases in [Ca²⁺]_i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK_2i4svR resensitized five times faster than CCK₂R. Without agonist, 80% of CCK_2i4svR is located in an intracellular compartment. In contrast, 80% of CCK₂R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK_2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK_2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK_2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.