从肺中溶解的血管活性肠肽受体的特性分析

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung.

作者信息

Paul S, Said S I

出版信息

J Biol Chem. 1987 Jan 5;262(1):158-62.

Abstract

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.

摘要

两性离子去污剂CHAPS用于溶解豚鼠肺中血管活性肠肽（VIP）的功能性受体。在3 mM CHAPS中通过高效凝胶过滤分离溶解的受体，得到两个活性组分，其表观斯托克斯半径分别为5.9±0.1和2.3±0.1 nm。125I-VIP与这两个受体组分的结合是时间依赖性、可逆且可饱和的。胰蛋白酶破坏了受体组分的结合活性，表明它们具有蛋白质性质。未标记的VIP竞争性地取代了125I-VIP与5.9 nm组分（IC50 = 240 pM）和2.3 nm组分（IC50 = 1.2 μM）的结合。Scatchard分析表明每个受体组分中存在单一类别的结合位点，5.9 nm和2.3 nm斯托克斯半径组分的Kd值分别为300 pM和0.97 μM。当高亲和力的5.9 nm斯托克斯半径组分在9 nM CHAPS中重新层析时，46%的结合活性在低亲和力的2.3 nm斯托克斯半径组分中洗脱，表明后者是高亲和力受体复合物解离的产物。GTP抑制125I-VIP与高亲和力复合物的结合，但不抑制低亲和力组分的结合。用GTP处理的高亲和力受体对VIP结合的Scatchard图表明存在两个不同的结合位点（Kd 4.4和153 nM），而未处理的受体中得到的是单一结合位点（Kd = 0.3 nM）。不可水解的GTP类似物鸟苷-5'-基-亚氨基二磷酸抑制高亲和力受体组分对VIP的结合，其效力与GTP几乎相当。这些观察结果表明，GTP结合调节蛋白在功能上与高亲和力受体复合物中的VIP结合亚基偶联。这两个受体组分的肽特异性特征不同。肽组氨酸异亮氨酸和生长激素释放因子，与VIP同源的肽，在取代高亲和力受体复合物对125I-VIP的结合方面，效力分别比VIP低87.5倍和22.9倍。另一方面，生长激素释放因子在取代低亲和力组分的结合方面比VIP更有效（22.7倍），而肽组氨酸异亮氨酸则比VIP效力低（31.3倍）。