alexa Evidence for multiple protein constituents of the somatostatin receptor in pituitary tumor cells: affinity cross-linking and molecular characterization.


Journal of Nuclear Medicine & Radiation Therapy

Author(s): Murthy KK, Srikant CB, Patel YC

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Abstract Previous studies have shown that somatostatin receptors on AtT-20 and GH3 pituitary tumor cells show relative preference for binding somatostatin-28 (S-28) and somatostatin-14 (S-14), respectively. Here we have attempted to determine whether this selectivity can be explained by molecular heterogeneity of the receptor. Cells were incubated with [125I-Tyr11]S-14, [125I-Leu8-D-Trp22,Tyr25]S-28, and [125I-Tyr3]SMS, and the bound ligand was chemically cross-linked with bis-[2-succinimido-oxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, or dithiobis (succinimidyl propionate). The solubilized cross-linked material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. [125I-Tyr11]S-14 labeled three specific receptor proteins of 57K, 42K, and 27K mol wt in AtT-20 cells. The relative proportions of the protein bands were unaltered by the use of whole cells or cell membranes or by the inclusion of dithiothreitol or antiproteolytic agents. With both [125I-Tyr11]S-14 and [125I-LTT]S-28, the 57K protein constituted the major labeled component, representing 70-75\% of the total cross-linked proteins. Labeling of the three protein species by [125I-Tyr11]S-14 and [125I-LTT]S-28 was inhibited by both S-14 and S-28 in a dose-dependent manner. S-28 was 10-20 times more potent than S-14 for inhibiting the labeling by both ligands of the principal receptor species of 57K. By contrast, when a radioiodinated derivative of the octapeptide analog octreotide ([125I-Tyr3]SMS) was used as ligand, the 27K protein was preferentially labeled, whereas the 57K and 42K bands were detected only as minor components. Labeling of GH3 cells with [125I-Tyr11]S-14 and [125I-LTT]S-28 revealed three cross-linked proteins of 57K, 42K, and 27K mol wt similar to those observed in AtT-20 cells. However, in this cell line the 27K protein, not the 57K species, was the dominant component identified with these two ligands, comprising 40-50\% of the total cross-linked proteins. These results suggest that there are three somatostatin receptor proteins of 57K, 42K, and 27K in pituitary cells. In AtT-20 cells, the 57K protein constitutes the major receptor protein labeled by [125I-Tyr11]S-14 and [125I-LTT]S-28, whereas the 27K protein is the major species labeled by [125I-Tyr3]SMS. The 27K, not the 57K, moiety is the principal receptor form in GH3 cells. Such ligand- and tissue-selective binding by the somatostatin receptor provides strong evidence for receptor molecular heterogeneity. This article was published in Endocrinology and referenced in Journal of Nuclear Medicine & Radiation Therapy

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