Binding and internalization of a iodinated substance P analog by cultured anterior pituitary cells.
Substance P binding to cultured anterior pituitary cells was characterized using Bolton-Hunter iodinated substance P as a ligand. At 0 degrees C, the interaction of the ligand and the cellular surface binding sites was found to be specific, rapid and reversible. Scatchard and Hill analysis of specific binding revealed a single class of non-interacting binding sites with a high affinity (KD = 0.48 nM) and a moderate density of binding sites (Bmax = 1187 binding sites/cell). At 37 degrees C a NaOH-soluble intracellular ligand pool was observed in addition to a surface-bound ligand pool released by a low pH buffer. Thus, substance P seems to be internalized after binding to cellular surface binding sites by means of receptor-mediated endocytosis. The internalization was rapid and could be blocked by colchicine (20 microM), an inhibitor of microtubuli assembly. Following internalization, intracellular degradation of the ligand could be demonstrated. Leupeptin (100 microM), an inhibitor of certain lysosomal enzymes could inhibit the cellular degradation of the added ligand, but had only a moderate influence on internalization. These results demonstrate that substance P after binding to a surface-localized receptor on its pituitary target cells is internalized and subsequently degraded by lysosomal enzymes.
Larsen
P J
PJ
Medical Institute of Anatomy B, University of Copenhagen, Denmark.
Mikkelsen
J D
JD
Mau
S
S
Saermark
T
T
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000032:analysis; D012972:Sodium Hydroxide; D013373:Substance P / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism*
C043194:Bolton Hunter reagent-substance P conjugate; D007457:Iodine Radioisotopes; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013388:Succinimides; D013373:Substance P; D012972:Sodium Hydroxide