Bhogal R, Smith D M, Purkiss P, Bloom S R
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Endocrinology. 1993 Nov;133(5):2351-61. doi: 10.1210/endo.133.5.8404688.
We have investigated the binding of rat [125I]islet amyloid polypeptide (IAPP) and [125I]calcitonin gene-related peptide (CGRP) to lung membranes from the rat, rabbit, and bull and have characterized the mol wt (M(r)) of the binding site for each ligand by chemical crosslinking. Results imply the existence of three distinct types of binding site demonstrated by both [125I]CGRP and [125I]IAPP in each of the three species investigated. These were differentiated by the relative potencies of displacement by rat CGRP, human CGRP-(8-37), rat IAPP, and the rat IAPP fragments IAPP-(8-37), IAPP-(12-37), IAPP-(25-37), and IAPP-(28-37). High affinity binding sites were identified for [125I]CGRP [rat Ki, 0.119 +/- 0.027 nM (n = 6); rabbit Ki, 0.944 +/- 0.075 nM (n = 6); bull Ki, 0.20 +/- 0.031 nM (n = 6)], and CGRP-(8-37) was found to displace [125I]CGRP in all species [rat Ki, 6.63 +/- 0.91 nM (n = 6); rabbit Ki, 22.70 +/- 3.79 nM (n = 6); bovine Ki, 26.9 +/- 0.21 nM (n = 3)]. Compared to CGRP-(8-37), displacement by IAPP also showed varying affinities that were similar to that of CGRP-(8-37) (rat), lower than that of CGRP-(8-37) (rabbit), or higher than that of CGRP-(8-37) (bull). Truncation of IAPP caused large parallel decreases in its affinity for [125I]CGRP in the rabbit and bull by the loss of residues 1-8 (rabbit) and 1-12 (bull), but was not as pronounced in the rat. [125I]IAPP demonstrated high affinity binding in each species [rat Ki, 5.86 +/- 0.86 nM (n = 6); rabbit Ki, 18.72 +/- 2.90 nM (n = 6); bull Ki, 1.97 +/- 0.40 nM (n = 6)]. Truncation of IAPP caused a reduction of its affinity for [125I]IAPP in all species by the loss of residues 1-28. Chemical cross-linking analysis indicated binding of both ligands to sites of 64,000 M(r) in the rat and 50,500 and 51,000 M(r) in the rabbit and bull, respectively. In addition, [125I]IAPP bound to to a site of 100,000 M(r) in the rat. [125I]CGRP and [125I]IAPP binding were reduced in the presence of guanosine 5-o-(3-Thiotriphosphate) in all species, indicating an association with G-proteins. This study implies the existence of CGRP/IAPP-binding sites in the lungs of these species that show varying and complex patterns of displacement by CGRP, IAPP, and their fragments.
我们研究了大鼠[125I]胰岛淀粉样多肽（IAPP）和[125I]降钙素基因相关肽（CGRP）与大鼠、兔和牛的肺膜的结合，并通过化学交联对每个配体结合位点的分子量（M(r)）进行了表征。结果表明，在所研究的三个物种中，[125I]CGRP和[125I]IAPP均显示存在三种不同类型的结合位点。这些结合位点可通过大鼠CGRP、人CGRP-(8 - 37)、大鼠IAPP以及大鼠IAPP片段IAPP-(8 - 37)、IAPP-(12 - 37)、IAPP-(25 - 37)和IAPP-(28 - 37)的相对置换效力来区分。已鉴定出[125I]CGRP的高亲和力结合位点[大鼠Ki，0.119±0.027 nM（n = 6）；兔Ki，0.944±0.075 nM（n = 6）；牛Ki，0.20±0.031 nM（n = 6）]，并且发现CGRP-(8 - 37)在所有物种中均可置换[125I]CGRP [大鼠Ki，6.63±0.91 nM（n = 6）；兔Ki，22.70±3.79 nM（n = 6）；牛Ki，26.9±0.21 nM（n = 3）]。与CGRP-(8 - 37)相比，IAPP的置换也显示出不同的亲和力，与CGRP-(8 - 37)相似（大鼠）、低于CGRP-(8 - 37)（兔）或高于CGRP-(8 - 37)（牛）。IAPP的截短导致其对兔和牛的[125I]CGRP的亲和力大幅平行下降，原因是分别缺失了1 - 8位残基（兔）和1 - 12位残基（牛），但在大鼠中不那么明显。[125I]IAPP在每个物种中均显示出高亲和力结合[大鼠Ki，5.86±0.86 nM（n = 6）；兔Ki，18.72±2.90 nM（n = 6）；牛Ki，1.97±0.40 nM（n = 6）]。IAPP的截短导致其在所有物种中因缺失1 - 28位残基而对[125I]IAPP的亲和力降低。化学交联分析表明，两种配体分别与大鼠中分子量为64,000 M(r)、兔和牛中分子量为50,500和51,000 M(r)的位点结合。此外，[125I]IAPP在大鼠中还与分子量为100,000 M(r)的位点结合。在所有物种中，鸟苷5 - o-(3 - 硫代三磷酸)存在时，[I25I]CGRP和[125I]IAPP的结合均减少，表明与G蛋白有关联。本研究表明，这些物种的肺中存在CGRP/IAPP结合位点，其被CGRP、IAPP及其片段置换的模式各不相同且复杂。
