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. 1995 Mar 2;128(6):1029–1041. doi: 10.1083/jcb.128.6.1029

Dual pathways of internalization of the cholecystokinin receptor

PMCID: PMC2120418  PMID: 7896869

Abstract

Receptor molecules play a major role in the desensitization of agonist- stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.

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Selected References

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  1. Abdelmoumene S., Gardner J. D. Cholecystokinin-induced desensitization of enzyme secretion in dispersed acini from guinea pig pancreas. Am J Physiol. 1980 Oct;239(4):G272–G279. doi: 10.1152/ajpgi.1980.239.4.G272. [DOI] [PubMed] [Google Scholar]
  2. Anderson K. M., Peach M. J. Receptor binding and internalization of a unique biologically active angiotensin II-colloidal gold conjugate: morphological analysis of angiotensin II processing in isolated vascular strips. J Vasc Res. 1994 Jan-Feb;31(1):10–17. doi: 10.1159/000159026. [DOI] [PubMed] [Google Scholar]
  3. Anderson R. G. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10909–10913. doi: 10.1073/pnas.90.23.10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson R. G. Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr Opin Cell Biol. 1993 Aug;5(4):647–652. doi: 10.1016/0955-0674(93)90135-d. [DOI] [PubMed] [Google Scholar]
  5. Barak L. S., Tiberi M., Freedman N. J., Kwatra M. M., Lefkowitz R. J., Caron M. G. A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration. J Biol Chem. 1994 Jan 28;269(4):2790–2795. [PubMed] [Google Scholar]
  6. Benovic J. L., Bouvier M., Caron M. G., Lefkowitz R. J. Regulation of adenylyl cyclase-coupled beta-adrenergic receptors. Annu Rev Cell Biol. 1988;4:405–428. doi: 10.1146/annurev.cb.04.110188.002201. [DOI] [PubMed] [Google Scholar]
  7. Boden P. R., Hill R. G. Effects of cholecystokinin and pentagastrin on rat hippocampal neurones maintained in vitro. Neuropeptides. 1988 Aug-Sep;12(2):95–103. doi: 10.1016/0143-4179(88)90037-6. [DOI] [PubMed] [Google Scholar]
  8. Cherner J. A., Naik L., Singh G. Cholecystokinin-induced desensitization of pepsinogen secretion from chief cells. Am J Physiol. 1989 May;256(5 Pt 1):G837–G845. doi: 10.1152/ajpgi.1989.256.5.G837. [DOI] [PubMed] [Google Scholar]
  9. Chun M., Liyanage U. K., Lisanti M. P., Lodish H. F. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11728–11732. doi: 10.1073/pnas.91.24.11728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daukas G., Zigmond S. H. Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes. J Cell Biol. 1985 Nov;101(5 Pt 1):1673–1679. doi: 10.1083/jcb.101.5.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Lean A., Stadel J. M., Lefkowitz R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem. 1980 Aug 10;255(15):7108–7117. [PubMed] [Google Scholar]
  12. Farese R. V., Orchard J. L., Larson R. E., Sabir M. A., Davis J. S. Phosphatidylinositol hydrolysis and phosphatidylinositol 4',5-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas. Biochim Biophys Acta. 1985 Aug 30;846(2):296–304. doi: 10.1016/0167-4889(85)90077-1. [DOI] [PubMed] [Google Scholar]
  13. Gates L. K., Ulrich C. D., Miller L. J. Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner. Am J Physiol. 1993 May;264(5 Pt 1):G840–G847. doi: 10.1152/ajpgi.1993.264.5.G840. [DOI] [PubMed] [Google Scholar]
  14. Ghinea N., Vu Hai M. T., Groyer-Picard M. T., Houllier A., Schoëvaërt D., Milgrom E. Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells. J Cell Biol. 1992 Sep;118(6):1347–1358. doi: 10.1083/jcb.118.6.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hansen S. H., Sandvig K., van Deurs B. Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J Cell Biol. 1993 Apr;121(1):61–72. doi: 10.1083/jcb.121.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hansen S. H., Sandvig K., van Deurs B. Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors. J Cell Biol. 1993 Oct;123(1):89–97. doi: 10.1083/jcb.123.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hausdorff W. P., Caron M. G., Lefkowitz R. J. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J. 1990 Aug;4(11):2881–2889. [PubMed] [Google Scholar]
  18. Heuser J. E., Anderson R. G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989 Feb;108(2):389–400. doi: 10.1083/jcb.108.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoxie J. A., Ahuja M., Belmonte E., Pizarro S., Parton R., Brass L. F. Internalization and recycling of activated thrombin receptors. J Biol Chem. 1993 Jun 25;268(18):13756–13763. [PubMed] [Google Scholar]
  20. Klueppelberg U. G., Gates L. K., Gorelick F. S., Miller L. J. Agonist-regulated phosphorylation of the pancreatic cholecystokinin receptor. J Biol Chem. 1991 Feb 5;266(4):2403–2408. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Larkin J. M., Brown M. S., Goldstein J. L., Anderson R. G. Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell. 1983 May;33(1):273–285. doi: 10.1016/0092-8674(83)90356-2. [DOI] [PubMed] [Google Scholar]
  23. Larkin J. M., Donzell W. C., Anderson R. G. Modulation of intracellular potassium and ATP: effects on coated pit function in fibroblasts and hepatocytes. J Cell Physiol. 1985 Sep;124(3):372–378. doi: 10.1002/jcp.1041240303. [DOI] [PubMed] [Google Scholar]
  24. Larkin J. M., Donzell W. C., Anderson R. G. Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices. J Cell Biol. 1986 Dec;103(6 Pt 2):2619–2627. doi: 10.1083/jcb.103.6.2619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lefkowitz R. J., Caron M. G. Regulation of adrenergic receptor function by phosphorylation. Curr Top Cell Regul. 1986;28:209–231. doi: 10.1016/b978-0-12-152828-7.50007-x. [DOI] [PubMed] [Google Scholar]
  26. Lefkowitz R. J., Cotecchia S., Kjelsberg M. A., Pitcher J., Koch W. J., Inglese J., Caron M. G. Adrenergic receptors: recent insights into their mechanism of activation and desensitization. Adv Second Messenger Phosphoprotein Res. 1993;28:1–9. [PubMed] [Google Scholar]
  27. Liggett S. B., Bouvier M., Hausdorff W. P., O'Dowd B., Caron M. G., Lefkowitz R. J. Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites. Mol Pharmacol. 1989 Oct;36(4):641–646. [PubMed] [Google Scholar]
  28. Lisanti M. P., Scherer P. E., Tang Z., Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 1994 Jul;4(7):231–235. doi: 10.1016/0962-8924(94)90114-7. [DOI] [PubMed] [Google Scholar]
  29. Lisanti M. P., Tang Z. L., Sargiacomo M. Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J Cell Biol. 1993 Nov;123(3):595–604. doi: 10.1083/jcb.123.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lohse M. J. Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta. 1993 Nov 7;1179(2):171–188. doi: 10.1016/0167-4889(93)90139-g. [DOI] [PubMed] [Google Scholar]
  31. Lutz M. P., Pinon D. I., Gates L. K., Shenolikar S., Miller L. J. Control of cholecystokinin receptor dephosphorylation in pancreatic acinar cells. J Biol Chem. 1993 Jun 5;268(16):12136–12142. [PubMed] [Google Scholar]
  32. Lutz M. P., Sutor S. L., Abraham R. T., Miller L. J. A role for cholecystokinin-stimulated protein tyrosine phosphorylation in regulated secretion by the pancreatic acinar cell. J Biol Chem. 1993 May 25;268(15):11119–11124. [PubMed] [Google Scholar]
  33. Miller L. J., Rosenzweig S. A., Jamieson J. D. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor, N alpha (125I-desaminotyrosyl)CCK-8, and its interactions with pancreatic acini. J Biol Chem. 1981 Dec 10;256(23):12417–12423. [PubMed] [Google Scholar]
  34. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  35. Ng G. Y., Mouillac B., George S. R., Caron M., Dennis M., Bouvier M., O'Dowd B. F. Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor. Eur J Pharmacol. 1994 Mar 15;267(1):7–19. doi: 10.1016/0922-4106(94)90219-4. [DOI] [PubMed] [Google Scholar]
  36. Otsuki M., Williams J. A. Amylase secretion by isolated pancreatic acini after chronic cholecystokinin treatment in vivo. Am J Physiol. 1983 Jun;244(6):G683–G688. doi: 10.1152/ajpgi.1983.244.6.G683. [DOI] [PubMed] [Google Scholar]
  37. Ozcelebi F., Miller L. J. Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells. J Biol Chem. 1995 Feb 17;270(7):3435–3441. doi: 10.1074/jbc.270.7.3435. [DOI] [PubMed] [Google Scholar]
  38. Papac D. I., Thornburg K. R., Büllesbach E. E., Crouch R. K., Knapp D. R. Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry. J Biol Chem. 1992 Aug 25;267(24):16889–16894. [PubMed] [Google Scholar]
  39. Powers S. P., Fourmy D., Gaisano H., Miller L. J. Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors. D-Tyr-Gly-[Nle28,31,pNO2-Phe33)CCK-26-33). J Biol Chem. 1988 Apr 15;263(11):5295–5300. [PubMed] [Google Scholar]
  40. Powers S. P., Pinon D. I., Miller L. J. Use of N,O-bis-Fmoc-D-Tyr-ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides. Int J Pept Protein Res. 1988 May;31(5):429–434. doi: 10.1111/j.1399-3011.1988.tb00899.x. [DOI] [PubMed] [Google Scholar]
  41. Raposo G., Dunia I., Delavier-Klutchko C., Kaveri S., Strosberg A. D., Benedetti E. L. Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles. Eur J Cell Biol. 1989 Dec;50(2):340–352. [PubMed] [Google Scholar]
  42. Sandvig K., van Deurs B. Endocytosis without clathrin. Trends Cell Biol. 1994 Aug;4(8):275–277. doi: 10.1016/0962-8924(94)90211-9. [DOI] [PubMed] [Google Scholar]
  43. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shinohara S., Kawasaki K. Desensitization of cholecystokininB receptors in GH3 cells. J Neurochem. 1994 Apr;62(4):1352–1356. doi: 10.1046/j.1471-4159.1994.62041352.x. [DOI] [PubMed] [Google Scholar]
  45. Steer M. L. Pathobiology of experimental acute pancreatitis. Yale J Biol Med. 1992 Sep-Oct;65(5):421–440. [PMC free article] [PubMed] [Google Scholar]
  46. Strosberg A. D. Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins. Eur J Biochem. 1991 Feb 26;196(1):1–10. doi: 10.1111/j.1432-1033.1991.tb15778.x. [DOI] [PubMed] [Google Scholar]
  47. Tam J. P. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5409–5413. doi: 10.1073/pnas.85.15.5409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  49. Trowbridge I. S. Endocytosis and signals for internalization. Curr Opin Cell Biol. 1991 Aug;3(4):634–641. doi: 10.1016/0955-0674(91)90034-v. [DOI] [PubMed] [Google Scholar]
  50. Ulrich C. D., 2nd, Pinon D. I., Hadac E. M., Holicky E. L., Chang-Miller A., Gates L. K., Miller L. J. Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors. Gastroenterology. 1993 Nov;105(5):1534–1543. doi: 10.1016/0016-5085(93)90162-6. [DOI] [PubMed] [Google Scholar]
  51. Yu S. S., Lefkowitz R. J., Hausdorff W. P. Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization. J Biol Chem. 1993 Jan 5;268(1):337–341. [PubMed] [Google Scholar]
  52. Zhu Z., Lutz M., Gates L. K., Miller L. J. Mechanisms of heterologous agonist-stimulated phosphorylation of cholecystokinin receptor. Am J Physiol. 1994 Apr;266(4 Pt 1):C904–C910. doi: 10.1152/ajpcell.1994.266.4.C904. [DOI] [PubMed] [Google Scholar]
  53. von Zastrow M., Kobilka B. K. Antagonist-dependent and -independent steps in the mechanism of adrenergic receptor internalization. J Biol Chem. 1994 Jul 15;269(28):18448–18452. [PubMed] [Google Scholar]
  54. von Zastrow M., Kobilka B. K. Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem. 1992 Feb 15;267(5):3530–3538. [PubMed] [Google Scholar]
  55. von Zastrow M., Link R., Daunt D., Barsh G., Kobilka B. Subtype-specific differences in the intracellular sorting of G protein-coupled receptors. J Biol Chem. 1993 Jan 15;268(2):763–766. [PubMed] [Google Scholar]

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