Glucagon-like peptide-1 (GLP-1) releases thyrotropin (TSH): Characterization of binding sites for GLP-1 on alpha-TSH cells

We have demonstrated specific binding sites for [I-125]glucagon-like peptide 1 (GLP-1) on membranes from the rodent thyrotrope cell line, alpha-TSH. Specific [I-125]GLP-1 binding was saturable and time dependent. Equilibrium saturation binding analysis was consistent with the presence of a single class of binding site (binding capacity, 85 +/- 7 fmol/mg protein) with a dissociation constant (K-d) of 28 +/- 13 pM. The specific GLP-1 receptor agonists, exendin-4 and exendin-3, and the antagonist, exendin-(9-39), bound to the receptor sites with high affinity (K-i = 190 +/- 70 pm: 130 +/- 50 and 1200 +/- 470 pM, respectively). Chemical cross-linking of [I-125]GLP-1-receptor complexes revealed a single band of 64,300 +/- 100 M(r) in alpha-TSH membranes. In addition, specific PCR studies demonstrated the presence of GLP-1 receptor messenger RNA. Binding of the peptide to alpha-TSH cell membranes resulted in increased intracellular cAMP concentrations (10 nM GLP-1, 1010 +/- 83 pmol/10(6) cells . h; control, 175 +/- 60 pmol/10(6) cells . h; P < 0.002), indicating that the receptor is linked to stimulation of adenylyl cyclase. GLP-1-mediated increases in cAMP were inhibited by exendin-(9-39) in a dose-dependent manner. Furthermore, GLP-1 stimulates basal TSH release from dispersed anterior pituitary cells in a concentration-dependent manner(100 nM GLP-1, 63 +/- 3 fmol/10 cells . h; control, 35 +/- 1 fmol/10(6) cells . h; P < 0.0005), but had no effect on basal PRL, GH, or LH release.