Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states.
Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.
Provow
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SA
Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA.
Veliçelebi
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G
D000818:Animals; D002268:Carboxypeptidases; D002918:Chymotrypsin; D004072:Digitonin; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008168:Lung / Q000032:analysis / Q000378:metabolism*; D008566:Membranes / Q000032:analysis / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013997:Time Factors; D014357:Trypsin
D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D011092:Polyethylene Glycols; D017830:Octoxynol; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D002918:Chymotrypsin; D014357:Trypsin; D004072:Digitonin