Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptor-activating peptides
Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptor-activating peptides
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DOI：
10.1139/cjpp-75-7-832
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发表时间：
1997-07-01
影响因子：
2.1
通讯作者：
Kawabata, A
中科院分区：
文献类型：
--
作者：
Hollenberg, MD;Saifeddine, M;Kawabata, A
我们使用了三种不同的生物测定系统(大鼠主动脉(RA)松弛；大鼠胃纵肌(LM)收缩；人胚胎肾脏293(HEK293)细胞钙信号)来评估来源于凝血酶受体(TRAPS，基于人类受体序列SFLLRNPNDK...)的受体激活肽类似物的活性和受体选择性。或蛋白水解酶激活受体2(PAR(2)APs，基于大鼠受体序列SLIGRL...)。我们的主要焦点是PAR(2)AP对PAR(2)的激活以及陷阱对PAR(2)的交叉激活。在RA和LM分析系统中，N-乙酰化(N-乙酰-SLIGRL-NH2)或具有反向N-末端序列(LSIGRL-NH2)的PAR(2)AP无论作为激动剂还是拮抗剂都是无效的。PAR第3位的丙氨酸取代AP(SLAGRL-NH2)导致生物活性显著降低，第1位的苏氨酸取代丝氨酸(TLIGRL-NH2)也是如此。然而，PAR(2)AP第4位的丙氨酸取代只引起活性的适度降低，导致了一种与人PAR(2)AP的效力相当的多肽(SLIARL-NH2)，SLIGKV-NH2。PAR(2)AP在RA、LM和HEK检测体系中的效价顺序为：SLIGRL-NH2>；SLIARL-NH2>；SLIGKV-NH2>；TLIGRL-NH2>；SLAGRL-NH2。在HEK细胞中，PAR(2)AP均未激活凝血酶受体(PAR(1))。然而，在HEK细胞实验中，TRAP，SFLLR-NH2，激活或脱敏PAR(1)和PAR(2)受体，而非洲爪哇TRAP，TFRIFD-NH2，选择性地激活或脱敏PAR(1)，而不是PAR(2)。通过构建人-非洲爪哇杂交肽，我们发现TRAP、TFLLR-NH2和SFLLFD-NH2选择性地激活HEK细胞中的凝血酶受体，而不激活或减敏PAR(2)。相反，陷阱SFLLRD-NH2和AFLLR-NH2激活或减敏PAR(1)和PAR(2)。在所有生物测定系统中，捕捉器的效力顺序为SFLLR-NH2类似或等于SFLLRD-NH2类似或等于TFLLR-NH2>；SFLLFD-NH2，TFRIFD-NH2。我们认为，PAR(2)AP的N-末端结构域以及位置3在PAR激活过程中起着重要作用。相反，TRAP基序中的第一和第五个氨基酸SFLLR-NH_1在激活凝血酶受体方面并不发挥独特的作用，但如果适当的修饰可以消除该肽交叉脱敏或激活PAR(2)的能力，从而对PAR(1)具有选择性。我们合成的PAR(1)和PAR(2)选择性多肽将用于评价PAR(1)和PAR(2)受体系统在体内的作用。
We have used three distinct bioassay systems (rat aorta (RA) relaxation; rat gastric longitudinal muscle (LM) contraction; human embryonic kidney 293 (HEK293) cell calcium signal) to evaluate the activity and receptor selectivity of analogues of the receptor-activating peptides derived either from the thrombin receptor (TRAPs, based on the human receptor sequence, SFLLRNPNDK...) or the proteinase-activated receptor 2 (PAR(2)APs, based on the rat receptor sequence SLIGRL...). Our main focus was on the activation of PAR(2) by PAR(2)APs and the cross-activation of PAR(2) by the TRAPs. In the RA and LM assay systems, PAR(2)APs that were either N-acetylated (N-acetyl-SLIGRL-NH2) or had a reverse N-terminal sequence (LSIGRL-NH2) were inactive, either as agonists or antagonists. An alanine substitution at position 3 of the PAR,AP (SLAGRL-NH2) led to a dramatic reduction of biological activity, as did substitution of threonine for serine at position 1 (TLIGRL-NH2). However, alanine substitution at PAR(2)AP position 4 caused only a modest reduction in activity, resulting in a peptide (SLIARL-NH2) with a potency equivalent to that of the human PAR(2)AP, SLIGKV-NH2. The order of potency of the PAR(2)APs in the RA, LM, and HEK assay systems was SLIGRL-NH2 > SLIARL-NH2>SLIGKV-NH2>TLIGRL-NH2> SLAGRL-NH2. In HEK cells, none of the PAR(2)APs activated the thrombin receptor (PAR(1)). However, in the HEK cell assay, the TRAP, SFLLR-NH2, activated or desensitized both PAR(1) and PAR(2) receptors, whereas the xenopus TRAP, TFRIFD-NH2, activated or desensitized selectively PAR(1) but not PAR(2). By constructing human-xenopus hybrid peptides, we found that the TRAPs, TFLLR-NH2, and SFLLFD-NH2 selectively activated the thrombin receptor in HEK cells without activating or desensitizing PAR(2). In contrast, the TRAPs SFLLRD-NH2 and AFLLR-NH2 activated or desensitized both PAR(1) and PAR(2). The order of potency for the TRAPs in all bioassay systems was SFLLR-NH2 similar or equal to SFLLRD-NH2 similar or equal to TFLLR-NH2 > SFLLFD-NH2, TFRIFD-NH2. We conclude that the N-terminal domain of the PAR(2)AP as well as positon 3 plays important roles for PAR, activation. Ln contrast, the first and fifth amino acids in the TRAP motif, SFLLR-NH,, do not play a unique role in activating the thrombin receptor, but if appropriately modified can abrogate the ability of this peptide to cross-desensitize or activate PAR(2), so as to be selective for PAR(1). The PAR(1)- and PAR(2)-selective peptides that we have synthesized will be of use for the evaluation of the roles after PAR(1) and PAR(2) receptor systems in vivo.