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In Vivo Phosphorylation of the Somatostatin 2A Receptor in Human Tumors

Department of Integrative Biology and Pharmacology (A.S., Q.L., Y.W.), University of Texas Health Science Center–Houston, Houston, Texas 77225; Department of Pharmacological and Pharmaceutical Sciences (B.J.K.), College of Pharmacy, University of Houston, Houston, Texas 77204; and Division of Cell Biology and Experimental Cancer Research (J.C.R.), Institute of Pathology, University of Berne, CH-3010 Berne, Switzerland

Address all correspondence and requests for reprints to: Dr. Agnes Schonbrunn, Department of Integrative Biology and Pharmacology, University of Texas–Houston, P.O. Box 20708, Houston, Texas 77225. Email: agnes.schonbrunn{at}uth.tmc.edu.

Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.

This investigation was supported, in part, by research grants from the National Institute of Arthritis, Diabetes, Digestive, and Kidney Disease (DK32234); by the Welch Foundation (AU-1410); and by the Texas Advanced Technology Program (Grant no. 011618-0032-2001).

Abbreviations: BAP, Bacterial alkaline phosphatase; C5a, complement 5a; DßM, dodecyl-ß-D-maltoside; GPCR, G protein-coupled receptor; PMSF, phenylmethylsulfonylfluoride; PNGase, peptide-N-glycosidase; SDS, sodium dodecyl sulfate; SS, somatostatin; sst, somatostatin receptor subtype; TACT, N,N',N'' triacetyl-chitotriose; TBS, Tris-buffered saline; TCA, trichloroacetic acid; WGA, wheat germ agglutinin.

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