METHODS

We have carried out extensive studies of receptor distribution in several species, including rat, pig, sheep, and ox (FIG. 3 ae). The richest source of CGRP receptors was in the pig cerebell~ m.~'-~~ Therefore, pig brains (-1000 brains) were used as the starting material for the isolation and purification of CGRP receptors. m Membrane preparation was as follows. Fresh pig brains were collected from an abattoir, transported on dry ice, and stored at-70" C until used. Cerebellar cortices were dissected on ice, and the tissues were homogenized in buffer containing 50 mmolil Tris HC1, 0.32 mmolil sucrose, 2 rnmolil PMFSA, and 200 KIUi ml aprotinin. Homogenate was then centrifuged at 1000 g for 10 minutes at 4" C, and the supernatant was recentrifuged at 30,000 g for 45 min at 4" C. After an extensive series of trials of various reagents (12 different detergents) and procedures for solubilization, 3-{3-(cholamidopropy1) dimethy1ammoniuo)-1-propane-sulfonate (CHAPS) and sodium cholate were identified as the two best candidates for solubilizing CGRP receptors from membranes. 60.6'In the presence of K+ and PO:-, at a critical protein: detergent ratio, this method allowed preferential solubilization of the membrane-bound receptors while retaining their binding activity to ['z51] CGRP. 60 A 30,000 g crude pellet was solubilized with 0.15% sodium cholate in 200 mmol/l phosphate buffer for 90 min at 4" C and recentrifuged at 45,000 g for 45 rnin. The protein concentration of the resuspended pellet was estimated by a dye-binding method using a microtiter plate reader. 50 FIGURE 4 shows displacement of [I2'I] CGRP from membrane-bound and solubilized receptors in the presence of increasing concentration of unlabeled CGRP. The affinity of [1Z51] CGRP to CGRP receptor was found to be lower after solubilization, in comparison to membranebound receptor.