Iodination of [Tyr11]somatostatin yields a super high affinity ligand for somatostatin receptors in GH4C1 pituitary cells

DH Presky and A Schonbrunn

Department of Pharmacology, Harvard Medical School, Boston, Massachusetts.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound [125I-Tyr1]SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I- tyrosine occurs concomitantly with the dissociation of [125I-Tyr1]SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of [125I- Tyr11]SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of [125I-Tyr11]SRIF (less than 50 pM) required 6 hr to reach equilibrium at 37 degrees compared with the 60 min required for [125I-Tyr1]SRIF. Analysis of the kinetics of [125I- Tyr11]SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for [125I- Tyr1]SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for [125I- Tyr11]SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for [125I-Tyr1] SRIF. In agreement with its much slower rate of dissociation, [125I-Tyr11]SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did [125I-Tyr1]SRIF (Kd = 350 pM). However, the apparent ED50 for [I-Tyr11]SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of [I-Tyr11]SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for [125I-Tyr1]SRIF, receptor-bound [125I-Tyr11]SRIF was not internalized and was released from the cells as a mixture of intact [125I-Tyr11]SRIF (30%) and the degradation product 125I-tyrosine (65%). Only approximately 5% of receptor-bound [125I-Tyr11]SRIF was released as a different degradation product. Our data demonstrate that [125I-Tyr11]SRIF is a better radioanalog than [125I-Tyr1]SRIF for binding studies with intact cells because of its higher affinity for the SRIF receptor. In addition, inasmuch as receptor-mediated degradation of bound ligand releases iodotyrosine from both position 1 and position 11 substituted analogs, aminopeptidases are unlikely to be entirely responsible for SRIF degradation. The superior binding properties of [125I-Tyr11]SRIF should facilitate the detection of SRIF receptors in other cell types.

Volume 34, Issue 5, pp. 651-658, 11/01/1988
Copyright © 1988 by American Society for Pharmacology and Experimental Therapeutics

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