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Mutational Analysis of Extracellular Cysteine Residues of Rat Secretin Receptor Shows that Disulfide Bridges are Essential for Receptor Function

  1. Jean-Pierre Vilardaga1,
  2. Emmanuel Di Paolo2,
  3. Christopher Bialek1,
  4. Philippe De Neef2,
  5. Magali Waelbroeck2,
  6. Alex Bollen1,
  7. Patrick Robberecht2,*

Article first published online: 16 JUL 2004

DOI: 10.1111/j.1432-1033.1997.00173.x

Issue

European Journal of Biochemistry

European Journal of Biochemistry

Volume 246, Issue 1, pages 173–180, May 1997

Additional Information

How to Cite

Vilardaga, J.-P., Di Paolo, E., Bialek, C., De Neef, P., Waelbroeck, M., Bollen, A. and Robberecht, P. (1997), Mutational Analysis of Extracellular Cysteine Residues of Rat Secretin Receptor Shows that Disulfide Bridges are Essential for Receptor Function. European Journal of Biochemistry, 246: 173–180. doi: 10.1111/j.1432-1033.1997.00173.x

Author Information

  1. 1

    Department of Applied Genetics, Faculty of Sciences, Université Libre de Bruxelles, Belgium

  2. 2

    Department of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Belgium

* P. Robberecht, Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Medeciné, Université Libre de Bruxelles, Bat. G/E, CP 611, 808 Route de Lennik, B-1070 Brussels, Belgium Fax: +32 2 555 62 30 E-mail: probbe@ulb.ac.be

Publication History

  1. Issue published online: 16 JUL 2004
  2. Article first published online: 16 JUL 2004
  3. (Received 3 February/17 March 1997) – EJB 97 0169/3

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Keywords:

  • secretin receptor;
  • cysteine;
  • point-mutation;
  • disulfide bridge

Abstract

  1. Top of page
  2. Abstract
  3. References

We attempted to express point-mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point-mutant receptors (C24→S, C44→S, C53→S, C67→S, C85→S and C101→S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point-mutant receptors (C11→S, C186→S, C193→S and C263→S) were able to bind poorly 125I-secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263→S), the four triple mutants, and through double mutants C186,193→S and Cl86,263→S with a very high (μM) EC30 value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11–C186, and between C193–C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11–C186 (but not the C193–C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.

Abbreviations.
CHO

Chinese hamster ovary

VIP

vasoactive intestinal polypeptide

References

  1. Top of page
  2. Abstract
  3. References
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