High affinity binding of 125I-neurotensin to dispersed cells from chicken liver and brain.
Dispersed cells from chicken brain and liver were found to possess cell surface binding sites for 125I-neurotensin (125I-NT). Scatchard analyses indicated the presence of high affinity (K4, 25-80 pM) and low affinity (Kd, 250-450 pM) components in adult tissues. Binding capacity was reduced 25-40% by incubation with pertussis toxin. Ontogenetic studies indicated that NT receptor capacity increased approximately 20-fold from the embryonic stage to adult. Cross-linking of 125I-NT to intact cells labeled one major band (52 kDa, > or = 90%) and two minor bands (40 and 90 kDa, < or = 10%) which could represent distinct NT-receptors or one receptor partly degraded or cross-linked to G-protein(s). The binding of 125I-NT to dispersed cells was enhanced by reduction with dithoithreitol and suppressed by alkylation with N-ethyl-maleimide (NEM), maleimidocaproic acid (MCA) and p-chloromercuribenzenesulfonate (PCMBS). Since MCA and PCMBS do not permeate cells, this suggests that the sulfhydryl group(s) critical to binding are located within the NT receptor itself. Preincubation of cells with NT prior to treatment with NEM diminished its inhibitory effect, suggesting that the critical SH-group(s) were within the NT binding pocket or were protected by an allosteric effect. These results suggest that one or more of the nine cysteine residues in the NT receptor is involved in the NT binding reaction.
Mitra
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SP
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655, USA.
Carraway
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RE
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000375:Aging / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D002208:Caproates / Q000494:pharmacology; D002642:Chick Embryo; D002645:Chickens; D017364:Collagenases; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D007457:Iodine Radioisotopes; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009496:Neurotensin / Q000378:metabolism*; D037342:Pertussis Toxin; D011869:Radioligand Assay; D018028:Receptors, Neurotensin / Q000378:metabolism*; D013696:Temperature; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002208:Caproates; D007457:Iodine Radioisotopes; D018028:Receptors, Neurotensin; D010566:Virulence Factors, Bordetella; D009496:Neurotensin; D002731:4-Chloromercuribenzenesulfonate; D037342:Pertussis Toxin; D017364:Collagenases; D005033:Ethylmaleimide; D004229:Dithiothreitol