Thyrotropin (TSH)-Releasing Hormone Regulation of TSH Subunit Biosynthesis and Glycosylation in Normal and Hypothyroid Rat Pituitaries*

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Abstract
The regulation of TSH apoprotein and carbohydrate biosynthesis by TRH was studied by incubating pituitaries from normal and hypothyroid (3 weeks postthyroidectomy) rats in medium containing varying doses of TRH, [14C] alanine or [35S]methionine, and [3H]glucosamine. Samples were sequentially treated with anti-TSHβ to precipitate TSH and free TSHβ, anti-LHβ to remove LH and free LHβ, and anti- LHα to precipitate free a-subunits. Total proteins were acid precipitated. All precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In hypothyroid samples, acute TRH (6 h) stimulated [3H] glucosamine incorporation into secreted combined α-subunit to 204% and secreted combined β-subunit to 227% of control values (P < 0.01), and stimulated [14C]alanine incorporation into secreted combined α-subunit to 201% and secreted combined β- subunit to 258% of control values (P < 0.01); pituitary content was not altered by TRH. In hypothyroid incubates, the halfmaximal response was 8 x 10−10 M TRH for both labeled precursors. In contrast, in normal samples, acute TRH (6 h) did not stimulate TSH subunit carbohydrate and apoprotein synthesis, but after 24 h, TRH stimulated [3H]glucosamine incorporation into both subunits of TSH to 270% of control values (P < 0.02), with no change in [14C]alanine incorporation. Free a-subunit synthesis was not altered by TRH in normal or hypothyroid incubates. The glucosamine to alanine ratio of total newly synthesized TSH, reflecting its relative glycosylation, was increased by TRH in both combined subunits in hypothyroid samples as early as 6 h (P < 0.05) and in normal samples only at 24 h (P < 0.01). In summary, 1) TRH in hypothyroid incubates stimulated apoprotein and carbohydrate synthesis in combined α- and β- subunits, but not free a-subunits, at 6 and 24 h. 2) In normal pituitary incubates, TRH stimulated TSH subunit carbohydrate, but not apoprotein, synthesis only at 24 h. 3) TRH increased the relative glycosylation of TSH in hypothyroid and normal rat pituitary incubates. Such alterations in TSH glycosylation may be due to structural changes in the carbohydrate moiety and may be important for hormone release, biological activity, or clearance. (Endocrinology116: 1968–1976, 1985)

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