Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Feb 15;90(4):1295–1299. doi: 10.1073/pnas.90.4.1295

Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.

B Schwaller 1, E Calef 1, C Gitler 1, K Rosenheck 1
PMCID: PMC45859  PMID: 8433989

Abstract

To investigate the involvement of cytosolic proteins in exocytosis, a system with high temporal and spatial resolution has been developed that allows us to detect the interaction of Ca(2+)- and membrane-binding proteins with the plasma membrane during stimulation of intact chromaffin and PC12 (rat pheochromocytoma) cells. We used 5-iodonaphthalene-1-azide (INA), a hydrophobic label that rapidly partitions into the lipid bilayer of biological membranes. Upon photolysis the label covalently attaches to membrane-embedded domains of proteins. Cells, preincubated with INA in the dark, were stimulated by either 300 microM carbamoylcholine or 60 mM K+ and irradiated (20 s) at various time intervals after stimulation. Subsequently, the cytosolic Ca(2+)- and membrane-binding proteins were isolated in the presence of EGTA (EGTA extract). Of the approximately 40 proteins in the EGTA extract, 15 (15-100 kDa) are labeled in both cell types. Upon stimulation, labeling is increased up to 3-fold in some of the proteins compared to cells labeled under basal conditions. In the absence of external Ca2+, no increase is observed. The rate of label incorporation is similar to the rate of exocytosis in several of these proteins. These results indicate that in the event of triggered exocytosis some of the Ca(2+)-binding proteins interact with the plasma membrane and temporarily embed in the lipid bilayer. Our findings support the hypothesis according to which stimulus-induced alterations in the structure of the Ca(2+)-binding proteins lead to their transient insertion into the membrane and thereby to membrane fusion.

Full text

PDF
1295

Images in this article

1296Image
on p.1296
1297Image
on p.1297
1298Image
on p.1298
1298Image
on p.1298

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahnert-Hilger G., Bader M. F., Bhakdi S., Gratzl M. Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion. J Neurochem. 1989 Jun;52(6):1751–1758. doi: 10.1111/j.1471-4159.1989.tb07253.x. [DOI] [PubMed] [Google Scholar]
  2. Amy C. M., Kirshner N. Phosphorylation of adrenal medulla cell proteins in conjunction with stimulation of catecholamine secretion. J Neurochem. 1981 Mar;36(3):847–854. doi: 10.1111/j.1471-4159.1981.tb01671.x. [DOI] [PubMed] [Google Scholar]
  3. Bercovici T., Gitler C. 5-[125I]Iodonaphthyl azide, a reagent to determine the penetration of proteins into the lipid bilayer of biological membranes. Biochemistry. 1978 Apr 18;17(8):1484–1489. doi: 10.1021/bi00601a020. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Brooks J. C., Treml S. Catecholamine secretion by chemically skinned cultured chromaffin cells. J Neurochem. 1983 Feb;40(2):468–473. doi: 10.1111/j.1471-4159.1983.tb11306.x. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Creutz C. E., Zaks W. J., Hamman H. C., Crane S., Martin W. H., Gould K. L., Oddie K. M., Parsons S. J. Identification of chromaffin granule-binding proteins. Relationship of the chromobindins to calelectrin, synhibin, and the tyrosine kinase substrates p35 and p36. J Biol Chem. 1987 Feb 5;262(4):1860–1868. [PubMed] [Google Scholar]
  8. Côté A., Doucet J. P., Trifaró J. M. Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation. Neuroscience. 1986 Oct;19(2):629–645. doi: 10.1016/0306-4522(86)90286-1. [DOI] [PubMed] [Google Scholar]
  9. DOUGLAS W. W., POISNER A. M. On the mode of action of acetylcholine in evoking adrenal medullary secretion: increased uptake of calcium during the secretory response. J Physiol. 1962 Aug;162:385–392. doi: 10.1113/jphysiol.1962.sp006940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunn L. A., Holz R. W. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4989–4993. [PubMed] [Google Scholar]
  11. Geisow M. J. Common domain structure of Ca2+ and lipid-binding proteins. FEBS Lett. 1986 Jul 14;203(1):99–103. doi: 10.1016/0014-5793(86)81445-4. [DOI] [PubMed] [Google Scholar]
  12. Gerke V., Weber K. Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin. EMBO J. 1984 Jan;3(1):227–233. doi: 10.1002/j.1460-2075.1984.tb01789.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glenney J. R., Jr, Tack B., Powell M. A. Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta. J Cell Biol. 1987 Mar;104(3):503–511. doi: 10.1083/jcb.104.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Greenberg A., Zinder O. Alpha- and beta-receptor control of catecholamine secretion from isolated adrenal medulla cells. Cell Tissue Res. 1982;226(3):655–665. doi: 10.1007/BF00214792. [DOI] [PubMed] [Google Scholar]
  15. Greene L. A., Rein G. Release of (3H)norepinephrine from a clonal line of pheochromocytoma cells (PC12) by nicotinic cholinergic stimulation. Brain Res. 1977 Dec 23;138(3):521–528. doi: 10.1016/0006-8993(77)90687-4. [DOI] [PubMed] [Google Scholar]
  16. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gutierrez L. M., Ballesta J. J., Hidalgo M. J., Gandia L., García A. G., Reig J. A. A two-dimensional electrophoresis study of phosphorylation and dephosphorylation of chromaffin cell proteins in response to a secretory stimulus. J Neurochem. 1988 Oct;51(4):1023–1030. doi: 10.1111/j.1471-4159.1988.tb03063.x. [DOI] [PubMed] [Google Scholar]
  18. Kao L. S., Schneider A. S. Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study. J Biol Chem. 1986 Apr 15;261(11):4881–4888. [PubMed] [Google Scholar]
  19. Khanna N. C., Helwig E. D., Ikebuchi N. W., Fitzpatrick S., Bajwa R., Waisman D. M. Purification and characterization of annexin proteins from bovine lung. Biochemistry. 1990 May 22;29(20):4852–4862. doi: 10.1021/bi00472a015. [DOI] [PubMed] [Google Scholar]
  20. Klee C. B. Ca2+-dependent phospholipid- (and membrane-) binding proteins. Biochemistry. 1988 Sep 6;27(18):6645–6653. doi: 10.1021/bi00418a001. [DOI] [PubMed] [Google Scholar]
  21. Klip A., Gitler C. Photoactive covalent labeling of membrane components from within the lipid core. Biochem Biophys Res Commun. 1974 Oct 8;60(3):1155–1162. doi: 10.1016/0006-291x(74)90433-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lee S. A., Holz R. W. Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol. J Biol Chem. 1986 Dec 25;261(36):17089–17098. [PubMed] [Google Scholar]
  24. Livneh E., Prywes R., Kashles O., Reiss N., Sasson I., Mory Y., Ullrich A., Schlessinger J. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J Biol Chem. 1986 Sep 25;261(27):12490–12497. [PubMed] [Google Scholar]
  25. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  26. Phillips J. H. Dynamic aspects of chromaffin granule structure. Neuroscience. 1982 Jul;7(7):1595–1609. doi: 10.1016/0306-4522(82)90017-3. [DOI] [PubMed] [Google Scholar]
  27. Roda L. G., Nolan J. A., Kim S. U., Hogue-Angeletti R. A. Isolation and characterization of chromaffin granules from a pheochromocytoma (PC 12) cell line. Exp Cell Res. 1980 Jul;128(1):103–109. doi: 10.1016/0014-4827(80)90392-4. [DOI] [PubMed] [Google Scholar]
  28. Smith A. D., Winkler H. Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. Biochem J. 1967 May;103(2):483–492. doi: 10.1042/bj1030483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wilson S. P., Kirshner N. Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4994–5000. [PubMed] [Google Scholar]
  30. Wilson S. P. Purification of adrenal chromaffin cells on Renografin gradients. J Neurosci Methods. 1987 Feb;19(2):163–171. doi: 10.1016/0165-0270(87)90031-8. [DOI] [PubMed] [Google Scholar]
  31. Winston V. Use of a polynomial exponential function to describe migration of proteins on sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis. 1989 Mar;10(3):220–222. doi: 10.1002/elps.1150100312. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES