Identification of two pairs of spatially approximated residues within the carboxyl terminus of secretin and its receptor
Identification of two pairs of spatially approximated residues within the carboxyl terminus of secretin and its receptor
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DOI：
10.1074/jbc.m000612200
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发表时间：
2000-08-25
影响因子：
4.8
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Dong, MQ;Asmann, YW;Miller, LJ
促胰液素家族多肽的羧基末端结构域已被证明包含与其受体高亲和力结合的关键决定因素。在这项工作中，我们研究了促胰液素中的羧基末端残基与原型促胰液素受体之间的相互作用。我们先前利用光亲和标记技术证明了促胰液素残基22与包括氨基末端前30个残基的受体结构域之间的空间近似(董，M.，Wang，Y.，Pinon，D.I.，Hadac，E.M.和Miller，L.J.(1999)J.Biol)。化学。274,903-909)，在此，我们进一步用对苯甲酰苯丙氨酸(BPA(22))探针标记受体残基Leu(17)，通过对野生型和突变型分泌素受体(V13M和V16M)的逐步切割和序列分析，我们还开发了一种新的探针，在促胰液素的第26位加入一个可发光的BPA，更靠近其羧基末端。该类似物也是一种有效的激动剂(EC50=72+/-6 pm)，并与促胰液素受体特异性结合，亲和力高(K-I=10.3+/-2.4 nM)。它以饱和的、特异的方式在单个位置共价标记促胰液素受体。通过化学和酶切割标记的野生型和A41M突变受体结构，以及免疫沉淀标记的受体片段，将其定位于Gly(34)和Ala(41)之间的残基片段。放射化学测序确定共价连接的位置为残基Leu(36)，这些新的见解，以及我们最近报告的6个残基在促胰液素的氨基末端半部分与该受体的这个相同的氨基末端区域之间的接触(DONG，M，，Wang，Y，Hadac，E，M.，Pinon，D，I.，Holky，E.L.和Miller，L.J.(1999)J.Biol)。化学。274,19161-19167)，支持这一区域的关键作用，使这种相互作用的分子细节引起了人们的主要兴趣。
The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909), Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant secretin receptors (V13M and V16M) and sequence analysis, We also developed a new probe incorporating a photolabile Bpa at position 26 of secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC50 = 72 +/- 6 pM) and bound to the secretin receptor specifically and with high affinity (K-i = 10.3 +/- 2.4 nM). It covalently labeled the secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36), These new insights, along with our recent report of contact between residue 6 within the aminoterminal half of secretin and this same amino-terminal region of this receptor (Dong, M,, Wang, Y,, Hadac, E, M., Pinon, D, I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.