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. 1984 Mar 1;98(3):1111–1118. doi: 10.1083/jcb.98.3.1111

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation

PMCID: PMC2113133  PMID: 6321516

Abstract

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid- villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF- cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

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Selected References

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  1. Abrahamson D. R., Rodewald R. Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine. J Cell Biol. 1981 Oct;91(1):270–280. doi: 10.1083/jcb.91.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahnen D. J., Santiago N. A., Cezard J. P., Gray G. M. Intestinal aminooligopeptidase. In vivo synthesis on intracellular membranes of rat jejunum. J Biol Chem. 1982 Oct 25;257(20):12129–12135. [PubMed] [Google Scholar]
  3. Allen R. H., Majerus P. W. Isolation of vitamin B12-binding proteins using affinity chromatography. 3. Purification and properties of human plasma transcobalamin II. J Biol Chem. 1972 Dec 10;247(23):7709–7717. [PubMed] [Google Scholar]
  4. Alpers D. H., Seetharam B. Pathophysiology of diseases involving intestinal brush-border proteins. N Engl J Med. 1977 May 5;296(18):1047–1050. doi: 10.1056/NEJM197705052961808. [DOI] [PubMed] [Google Scholar]
  5. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  6. Blok J., Mulder-Stapel A. A., Ginsel L. A., Daems W. T. Endocytosis in absorptive cells of cultured human small-intestinal tissue: horseradish peroxidase, lactoperoxidase, and ferritin as markers. Cell Tissue Res. 1981;216(1):1–13. doi: 10.1007/BF00234540. [DOI] [PubMed] [Google Scholar]
  7. Burger R. L., Schneider R. J., Mehlman C. S., Allen R. H. Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12. J Biol Chem. 1975 Oct 10;250(19):7707–7713. [PubMed] [Google Scholar]
  8. Cooper B. A. Complex of intrinsic factor and B12 in human ileum during vitamin B12 absorption. Am J Physiol. 1968 Apr;214(4):832–835. doi: 10.1152/ajplegacy.1968.214.4.832. [DOI] [PubMed] [Google Scholar]
  9. Donaldson R. M., Jr, Mackenzie I. L., Trier J. S. Intrinsic factor-mediated attachment of vitamin B12 to brush borders and microvillous membranes of hamster intestine. J Clin Invest. 1967 Jul;46(7):1215–1228. doi: 10.1172/JCI105615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Friend D. S., Farquhar M. G. Functions of coated vesicles during protein absorption in the rat vas deferens. J Cell Biol. 1967 Nov;35(2):357–376. doi: 10.1083/jcb.35.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HERBERT V., CASTLE W. B. Divalent cation and pH dependence of rat intrinsic factor action in everted sacs and mucosal homogenates of rat small intestine. J Clin Invest. 1961 Nov;40:1978–1983. doi: 10.1172/JCI104423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hauri H. P., Quaroni A., Isselbacher K. J. Biogenesis of intestinal plasma membrane: posttranslational route and cleavage of sucrase-isomaltase. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5183–5186. doi: 10.1073/pnas.76.10.5183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hines J. D., Rosenberg A., Harris J. W. Intrinsic factor-mediated radio-B12 uptake in sequential incubation studies using everted sacs of guinea pig small intestine: evidence that IF in not absorbed into the intestinal cell. Proc Soc Exp Biol Med. 1968 Nov;129(2):653–658. doi: 10.3181/00379727-129-33390. [DOI] [PubMed] [Google Scholar]
  15. Hooper D. C., Alpers D. H., Burger R. L., Mehlman C. S., Allen R. H. Characterization of ileal vitamin B12 Binding using homogeneous human and hog intrinsic factors. J Clin Invest. 1973 Dec;52(12):3074–3083. doi: 10.1172/JCI107506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levine J. S., Nakane P. K., Allen R. H. Human intrinsic factor secretion: immunocytochemical demonstration of membrane-associated vesicular transport in parietal cells. J Cell Biol. 1981 Sep;90(3):644–655. doi: 10.1083/jcb.90.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levine J. S., Nakane P. K., Allen R. H. Immunocytochemical localization of human intrinsic factor: the nonstimulated stomach. Gastroenterology. 1980 Sep;79(3):493–502. [PubMed] [Google Scholar]
  18. Levine J. S., Nakane P. K., Allen R. H. Immunocytochemical localization of intrinsic factor--cobalamin bound to the guinea pig ileum in vivo. Gastroenterology. 1982 Feb;82(2):284–290. [PubMed] [Google Scholar]
  19. Mircheff A. K., Sachs G., Hanna S. D., Labiner C. S., Rabon E., Douglas A. P., Walling M. W., Wright E. M. Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity. J Membr Biol. 1979 Nov 30;50(3-4):343–363. doi: 10.1007/BF01868897. [DOI] [PubMed] [Google Scholar]
  20. Renston R. H., Maloney D. G., Jones A. L., Hradek G. T., Wong K. Y., Goldfine I. D. Bile secretory apparatus: evidence for a vesicular transport mechanism for proteins in the rat, using horseradish peroxidase and [125I]insulin. Gastroenterology. 1980 Jun;78(6):1373–1388. [PubMed] [Google Scholar]
  21. Rothenberg S. P. Identification of a macromolecular factor in the ileum which binds intrinsic factor and immunologic identification of intrinsic factor in ileal extracts. J Clin Invest. 1968 Apr;47(4):913–923. doi: 10.1172/JCI105783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seetharam B., Alpers D. H., Allen R. H. Isolation and characterization of the ileal receptor for intrinsic factor-cobalamin. J Biol Chem. 1981 Apr 25;256(8):3785–3790. [PubMed] [Google Scholar]
  23. Seetharam B., Bagur S. S., Alpers D. H. Interaction of receptor for intrinsic factor-cobalamin complex with synthetic and brush-border lipids. J Biol Chem. 1981 Oct 10;256(19):9813–9815. [PubMed] [Google Scholar]
  24. Seetharam B., Bagur S. S., Alpers D. H. Isolation and characterization of proteolytically derived ileal receptor for intrinsic factor-cobalamin. J Biol Chem. 1982 Jan 10;257(1):183–189. [PubMed] [Google Scholar]
  25. Stefanini M., De Martino C., Zamboni L. Fixation of ejaculated spermatozoa for electron microscopy. Nature. 1967 Oct 14;216(5111):173–174. doi: 10.1038/216173a0. [DOI] [PubMed] [Google Scholar]
  26. Stockert R. J., Haimes H. B., Morell A. G., Novikoff P. M., Novikoff A. B., Quintana N., Sternlieb I. Endocytosis of asialoglycoprotein-enzyme conjugates by hepatocytes. Lab Invest. 1980 Dec;43(6):556–563. [PubMed] [Google Scholar]
  27. Willingham M. C., Maxfield F. R., Pastan I. H. alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions. J Cell Biol. 1979 Sep;82(3):614–625. doi: 10.1083/jcb.82.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Youngdahl-Turner P., Rosenberg L. E., Allen R. H. Binding and uptake of transcobalamin II by human fibroblasts. J Clin Invest. 1978 Jan;61(1):133–141. doi: 10.1172/JCI108911. [DOI] [PMC free article] [PubMed] [Google Scholar]

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