HIGH-AFFINITY BINDING OF I-125 NEUROTENSIN TO DISPERSED CELLS FROM CHICKEN LIVER AND BRAIN

Dispersed cells from chicken brain and liver were found to possess cel l surface binding sites for I-125-neurotensin (I-125-NT). Scatchard an alyses indicated the presence of high affinity (K-d, 25-80 pM) and low affinity (K-d, 250-450 pM) components in adult tissues. Binding capac ity was reduced 25-40% by incubation with pertussis toxin. Ontogenetic studies indicated that NT receptor capacity increased similar to 20-f old from the embryonic stage to adult. Cross-linking of I-125-NT to in tact cells labeled one major band (52 kDa, greater than or equal to 90 %) and two minor bands (40 and 90 kDa, less than or equal to 10%) whic h could represent distinct NT-receptors or one receptor partly degrade d or cross-linked to G-protein(s). The binding of I-125-NT to disperse d cells was enhanced by reduction with dithoithreitol and suppressed b y alkylation with N-ethyl-maleimide (NEM), maleimidocaproic acid (MCA) and p-chlornmercuribenzenesulfonate (PCMBS). Since MCA and PCMBS do n ot permeate cells, this suggests that the sulfhydryl group(s) critical to binding are located within the NT receptor itself. Preincubation o f cells with NT prior to treatment with NEM diminished its inhibitory effect, suggesting that the critical SH-group(s) were within the NT bi nding pocket or were protected by an allosteric effect. These results suggest that one or more of the nine cysteine residues in the NT recep tor is involved in the NT binding reaction. (C) 1997 Elsevier Science Inc.