Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog.
We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.
Brown
P J
PJ
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Lee
A B
AB
Norman
M G
MG
Presky
D H
DH
Schonbrunn
A
A
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D010179:Pancreas; D037342:Pertussis Toxin; D010911:Pituitary Neoplasms; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000345:Affinity Labels; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D010566:Virulence Factors, Bordetella; C065966:somatostatin, azidonitrobenzoyl-Lys(4)-iodo-Tyr(11)-; C065968:somatostatin, azidonitrobenzoyl-Lys(9)-iodo-Tyr(11)-; D006165:Guanylyl Imidodiphosphate; D013004:Somatostatin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins