Intestinal VIP receptors: differential effect of trypsin on the high and low affinity binding sites

The effects of trypsin treatment on VIP binding to rat intestinal epithelial cell membranes were examined. The decrease in specific binding of [125I]VIP is dependent on the amount of trypsin used and digestion time. Specific binding decreases by 50% after 8 min with 20 micrograms/ml trypsin. Trypsin is active in the 1-100 micrograms/ml concentration range (ED50 approximately equal to 5 micrograms/ml). Non-specific binding is unaltered by the enzyme. The effect of trypsin is abolished by trypsin inhibitor. Scatchard analysis of VIP binding reveals two types of binding sites: sites I characterized by a high affinity, a low capacity and a high sensitivity to low trypsin levels (1-5 micrograms/ml); sites II characterized by a low affinity, a high capacity, resistant to low trypsin levels (1-5 micrograms/ml) but sensitive to a high trypsin level (20 micrograms/ml). Trypsin decreases the binding capacity by lowering the site number without altering their affinity. Sites not destroyed by trypsin retain their functional characteristics: KD, sensitivity to GTP and coupling with adenylate cyclase. It is concluded that sites I and II are proteins with different structures and/or differently localized in the membrane.