Endocrinology, Vol 132, 146-154, Copyright © 1993 by Endocrine Society

ARTICLES

Characterization of a biotinylated somatostatin analog as a receptor probe

A Schonbrunn, AB Lee and PJ Brown
Department of Pharmacology, University of Texas Medical School, Houston 77225.

The neuropeptide somatostatin (SRIF) triggers its biological effects by binding to high affinity membrane receptors. To develop a ligand useful for receptor affinity purification and localization, we have examined the ability of a novel monobiotinylated SRIF derivative to bind to receptors and streptavidin. Unlabeled [N-Biotinyl, Leu8, D-Trp22, Tyr25]SRIF28 (Bio-SRIF28) competed for [125I-Tyr11]SRIF binding to GH4C1 pituitary cell membranes with a Ki of 337 +/- 95 pM, comparable to that of native SRIF (193 +/- 16 pM). Studies using HPLC purified [125I]Bio-SRIF28 showed that equilibrium binding to membranes occurred within 120 min at 30 C and that the peptide-receptor complex dissociated slowly (t1/2 = 4.7 h). Analysis of saturation binding data gave an equilibrium dissociation constant for [125I]Bio-SRIF28 of 66 +/- 20 pM. Photoaffinity cross-linking of [125I]Bio-SRIF28 to membranes covalently labeled a broad 85 kDa band, as previously reported with the photolabile SRIF analog, [125I-Tyr11, Azidonitrobenzoyl-Lys4]SRIF. The binding of [125I]Bio-SRIF28 was potently inhibited by SRIF (Ki = 171 +/- 36 pM) and SRIF28 (299 +/- 102 pM) but not by structurally unrelated peptides. Furthermore, [125I]Bio-SRIF28 did not bind to membranes from GH(1)2C1 pituitary cells, which do not respond to SRIF and which lack [125I-Tyr11]SRIF binding sites. Finally, GppNHp and GTP gamma S both decreased [125I]Bio-SRIF28 binding whereas AppNHp did not. These studies showed that [125I]Bio-SRIF28 bound with high affinity to specific, G-protein coupled SRIF receptors. [125I]Bio-SRIF28 also bound with high affinity to streptavidin and this binding was very stable (t1/2 for dissociation = 19 h). Therefore, the affinity of the receptor for the Bio-SRIF28-streptavidin complex was determined by measuring the potency with which this preformed complex competed for [125I-Tyr11]SRIF binding. The Ki of the Bio-SRIF28-streptavidin complex (1110 +/- 47 pM) was only 3 times higher than that of uncomplexed Bio-SRIF28 (Ki = 337 +/- 95 pM). Dissociation of the [125I]Bio-SRIF28-streptavidin complex from receptors was slow (t1/2 = 3.9 h) but was increased over 200-fold by 1 microM GTP gamma S (t1/2 < 1 min). These data show that Bio-SRIF28 was able to bind simultaneously and with high affinity both to SRIF receptors and to streptavidin to form a stable ternary complex. Further, receptor binding of the Bio-SRIF28-streptavidin complex could be regulated by the addition of guanine nucleotides. Thus, Bio-SRIF28 should be useful for the affinity purification and in situ localization of SRIF receptors.

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