Somatostatin receptor subtypes in the clonal anterior pituitary cell lines AtT-20 and GH3

The functional and biochemical characteristics of somatostatin (somatotropin release-inhibiting factor) (SRIF) receptor subtypes were examined in the clonal pituitary cell lines AtT-20 and GH3. SRIF inhibits evoked calcium influx into each of these cell lines. The rank order of potencies of structural analogues of SRIF to inhibit calcium influx into GH3 versus AtT-20 cells was different. Inhibitory actions of SRIF on calcium influx desensitized in AtT-20 cells but not GH3 cells. The biochemical properties of the SRIF receptor subtypes in AtT-20 and GH3 cells were assessed by photoaffinity labeling of each receptor with the nonreducible SRIF analogue [125I]CGP 23996 and the photocrosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. The covalently labeled receptors in both cell lines had the same size, 55 +/- 5 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalent binding of [125I]CGP-23996 to GH3 and AtT-20 cell membranes was blocked by 1 microM SRIF, somatostatin 28, Trp8-SRIF and was GTP sensitive. Analysis of the labeled receptors in GH3 and AtT-20 cell membranes by two-dimensional polyacrylamide gel electrophoresis indicated that they were of similar charge (pI = 6-6.5) and that they comigrate when applied together. Proteolysis of the GH3 and AtT-20 cell SRIF receptors with Staphylococcus aureus V-8 and thermolysin revealed similar peptide maps. Pretreatment of AtT-20 cells with different stable SRIF analogues abolished the subsequent equilibrium or covalent labeling of the SRIF receptor with [125I]CGP-23996. Similar treatment of GH3 cells did not reduce the covalent labeling of the SRIF receptor by [125I]CGP 23996. These studies indicate that the functional characteristics of SRIF receptors in GH3 and AtT-20 cells are different. However, clear differences in the biochemical properties of these receptor subtypes were not observed. Subtle variations in the structure of the SRIF receptors may therefore be responsible for the functional differences.