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 CCK2i4svR 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 CCK2i4svR desensitization, internalization, and resensitization. Gastrin1–17 (G17) binds to both CCK2R and CCK2i4svR 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, CCK2i4svR resensitized five times faster than CCK2R. Without agonist, 80% of CCK2i4svR 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 CCK2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.