IDENTIFICATION OF A DOMAIN IN THE CARBOXY-TERMINUS OF CCK RECEPTOR THAT AFFECTS ITS INTRACELLULAR TRAFFICKING

The carboxyterminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)(2-3)Y motif, a site of fatty acid acylation, and seri ne-and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefo re, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese ham ster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. E ach construct was synthesized and transported appropriately to the cel l surface, where CCK bound with high affinity, elicited an inositol 1, 4,5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these establ ished sequence motifs had no substantial effect on any of these parame ters of receptor and cellular function. However, an additional constru ct that truncated the carboxy terminus, eliminating an additional 15-a mino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other pa rameters of receptor function normal. This mutant receptor construct w as delayed at the stage of early endosomes, delaying its progress to t he lysosome-enriched perinuclear compartment from the rapid time cours e followed by wildtype receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new mot if important for intracellular receptor trafficking.