Characterization of a biotinylated somatostatin analog as a receptor probe.
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.
Schonbrunn
A
A
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Lee
A B
AB
Brown
P J
PJ
D000345:Affinity Labels; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000378:metabolism*; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000378:metabolism; D053561:Somatostatin-28 / Q000031:analogs & derivatives; D019809:Streptavidin
D000345:Affinity Labels; D001426:Bacterial Proteins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D017481:Receptors, Somatostatin; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D001710:Biotin; D053561:Somatostatin-28; C045259:somatostatin 28, Leu(8)-Trp(22)-iodo-Tyr(25)-; D019809:Streptavidin