Direct visualization of receptors for thyrotropin-releasing hormone with a fluorescein-labeled analog.

Abstract

Thyrotropin-releasing hormone (TRH) binds to specific receptors on GH4C1 rat pituitary tumor cells. A fluorescently-labeled analog of TRH was synthesized by coupling pGlu-His-ProNH(CH2)6NH2 to fluorescein isothiocyanate. The fluorescein-labeled peptide (FL-TRH) stimulated prolactin synthesis and release by GH4C1 cultures and bound to TRH receptors with an apparent Kd of 400 nM. Binding of FL-TRH to unfixed, viable GH4C1 cells was followed by fluorescence microscopy. After incubation with 1.4 .mu.M FL-TRH for 1 h at 37.degree. C, the surface of all cells was fluorescent and patches of intense fluorescence were evident. Control cultures incubated with FL-TRH and excess TRH were not fluorescent, and a line of rat pituitary tumor cells which lacks TRH receptors GH-Y cells displayed little fluorescence after incubation with FL-TRH. When GH4C1 cells were incubated with FL-TRH for 1 h at 37.degree. C and then with excess TRH for an additional 1 h, the fluorescence associated with the cells was diminished to control levels. The fluorescein-labeled peptide evidently labels specific TRH receptors.