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(2i4sv)R 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(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(2i4sv)R 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(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R 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(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.
Chao
Celia
C
Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Ives
Kirk L
KL
Goluszko
Elizabeth
E
Kolokoltsov
Andrey A
AA
Davey
Robert A
RA
Townsend
Courtney M
CM
Hellmich
Mark R
MR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001569:Benzodiazepines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D005456:Fluorescent Dyes; D005755:Gastrins / Q000378:metabolism / Q000494:pharmacology; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007438:Introns; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D043887:Receptor, Cholecystokinin B / Q000819:agonists / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D005755:Gastrins; D006727:Hormone Antagonists; D043887:Receptor, Cholecystokinin B; D001569:Benzodiazepines; C087260:YM 022; D049452:Green Fluorescent Proteins; C024286:3,3'-dioctadecylindocarbocyanine; C013550:gastrin 17; D019061:src-Family Kinases; D002118:Calcium