Journal of Biological Chemistry
Volume 272, Issue 38, 19 September 1997, Pages 23769-23774
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CELL BIOLOGY AND METABOLISM
Phosphorylation of Four Amino Acid Residues in the Carboxyl Terminus of the Rat Somatostatin Receptor Subtype 3 Is Crucial for Its Desensitization and Internalization*

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Agonist-dependent internalization of the rat somatostatin receptor subtype 3 (SSTR3) requires four hydroxyl amino acids (Ser341, Ser346, Ser351, and Thr357) in the receptor C terminus (Roth, A., Kreienkamp, H.-J., Nehring, R., Roostermann, D., Meyerhof, W. and Richter, D. (1997) DNA Cell Biol. 16, 111–119). Here we report on the molecular mechanism responsible for the endocytotic process by analyzing the agonist-dependent phosphorylation of wild-type and mutant receptors expressed in human embryonic kidney cells. Wild-type SSTR3 is phosphorylated in response to agonist treatment. Phosphorylation is markedly reduced in a S341A/S346A/S351A triple mutant and is also reduced, but to a lesser extent, in the T357A point mutant. Internalization of the wild-type receptor is preceded by a functional desensitization of the receptor; in contrast, the triple serine mutant does not desensitize after treatment with agonists as assayed by its ability to inhibit forskolin-stimulated adenylate cyclase activity. After internalization via a clathrin-coated vesicle mediated endocytotic pathway, SSTR3 efficiently recycles to the cell surface, suggesting that agonist mediated endocytosis is necessary for the functional resensitization of a phosphorylated and desensitized receptor.

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*

This work was supported in part by the Deutsche Forschungsgemeinschaft SFB232/B4 (to D. R. and W. M.) and Ri 192/20-1 (to D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A. R. and H.-J. K. contributed equally to this work.

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Submitted to fulfill the requirements for a Dr. rer. nat. at the Universität Hamburg.