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

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.