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 [125I]glucagon-like peptide 1 (GLP-1) on membranes from the rodent thyrotrope cell line, alpha-TSH. Specific [125I]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 (Kd) 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 (Ki = 190 +/- 70 pM; 130 +/- 50 and 1200 +/- 470 pM, respectively). Chemical cross-linking of [125I]GLP-1-receptor complexes revealed a single band of 64,300 +/- 100 Mr 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(6) cells.h; control, 35 +/- 1 fmol/10(6) cells.h; P < 0.0005), but had no effect on basal PRL, GH, or LH release.
Beak
S A
SA
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Small
C J
CJ
Ilovaiskaia
I
I
Hurley
J D
JD
Ghatei
M A
MA
Bloom
S R
SR
Smith
D M
DM
D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D029742:Caenorhabditis elegans Proteins; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D051880:Receptors, Notch; D013972:Thyrotropin / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D003432:Cross-Linking Reagents; C060620:Glp-1 protein, C elegans; D008562:Membrane Glycoproteins; D051880:Receptors, Notch; D013972:Thyrotropin; D000242:Cyclic AMP