GH4 pituitary cell variants selected as nonresponsive to thyrotropin-releasing hormone-enhanced substratum adhesion are nonresponsive to epidermal growth factor: Evidence for a common signaling defect

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Abstract
Thyrotropin-releasing hormone (TRH) and epidermal growth factor both enhance prolactin synthesis and substrate adhesion (a morphological change called stretching) of GH4 rat pituitary cells. We have examined TRH- and EGF-induced cell stretching using genetic and pharmacologic approaches. We selected and isolated a series of GH4 cell variants nonresponsive to TRH-induced cell stretching (str−). This selection yielded several variants that were nonresponsive to both TRH- and EGF-induced stretching but were still responsive to stretching induced by several other agents (tetradecanoylphorbol acetate [TPA], butyrate, and Nepla-nocin A). One of the str− variants (a14) was examined in detail. TRH, EGF, and TPA each enhanced prolactin synthesis in a14 cells, indicating that the a14 variant contained functional receptor binding sites for all 3 ligands as well as the capacity to generate those intracellular signals required for enhanced prolactin synthesis. Because the str− variants were isolated without selective pressure for EGF-induced stretching and because the possibility of more than one selectable mutation in all the variants is unlikely, we suggest that TRH and EGF share a common mechanism to induce cell stretching. We next examined whether the str− variants had a defect in a signaling pathway or in the biochemical endpoint for TRH- and EGF-induced cell stretching. A pharmacologic approach was utilized to investigate the biochemical basis for induced cell stretching. A synthetic Arg-Gly-Asp-Ser tetrapeptide (RGDS), specific for fibronectin and vitronectin adhesion receptors, inhibited TRH-, EGF-, and TPA-induced GH4 cell stretching and attachment to fibronectin- and vitronectin-coated dishes. These results suggest that the interaction between fibronectin and/or vitronectin and their receptor(s) may be a biochemical endpoint by which several agonists induce stretching of GH4 cells. Because the str− variant has RGDS-specific binding sites for fibronectin and vitronectin and responds to some agents that induce cell stretching via an RGDS receptor, we conclude that the a14 str− variant has a defect in an intracellular signaling pathway, shared by TRH and EGF, which induces cell stretching.