Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

The conserved NPXnY motif present in the gastrin-releasing peptide receptor is not a general sequestration sequence.

Exposure of the gastrin-releasing peptide (GRP) receptor to agonists causes a rapid desensitization of the receptor-stimulated mobilization of intracellular calcium. Homologous desensitization occurs by uncoupling the G-proteins from the receptor and by ligand induced internalization. The molecular determinants of desensitization of the GRP receptor are not well known. The importance of tyrosine 324 which is located in a highly conserved NPX2-3 Y motif of the GRP receptor was investigated. Kirsten murine sarcoma virus-transformed rat kidney (KNRK) cells were transfected with expression vectors encoding either the wild type or the mutant (tyrosine 324 to alanine 324) rat GRP receptor. The wild type and mutant GRP receptors were expressed at a high level in the KNRK cells, 2.0 x 10(6) and 0.5 x 10(6) receptors per cell, respectively. The wild type and mutant GRP receptors bound GRP with the same affinity (Kd = 6-7 nM). KNRK cells expressing the wild type or mutant GRP receptor had similar [Ca2+]i, dose response to GRP. KNRK cells expressing the GRP receptor rapidly internalized bound 125I-GRP at 37 degree C. Internalization was inhibited at 4 degrees C and by 0.45 m sucrose. The internalization of bound 125I-GRP by the mutant GRP receptor was identical to the wild type receptor. Fluorescent microscopy was used to directly observe the GRP receptor expressed on the surface of the KNRK cells and to visualize its ligand induced internalization. There was no difference in the pattern of internalization between the wild type and mutant GRP receptors expressed in KNRK cells. Therefore, the highly conserved tyrosine 324 does not have a role in GRP binding, receptor-G-protein interaction, or initial events of ligand induced receptor internalization. The NPXnY motif is not a general sequestration sequence for seven transmembrane G-protein linked receptors.