Mitselos Anna, Depoortere Inge, Peeters Theo L
Centre for Gastroenterological Research, Catholic University of Leuven, B-3000 Leuven, Belgium.
Biochem Pharmacol. 2007 Jan 1;73(1):115-24. doi: 10.1016/j.bcp.2006.09.011. Epub 2006 Sep 16.
Studies with fragments of the gastrointestinal peptide, motilin, indicate that the C-terminal region of this peptide plays an important role in the desensitization of the motilin receptor (MTLR).
To verify this hypothesis we studied the desensitization, phosphorylation and internalization induced by motilin analogues of different chain length with agonistic and antagonistic properties in CHO-MTLR cells.
We studied motilin [1-22], the [1-14] fragment, the analogues Phe(3)[1-22] and Phe(3)[1-14], and two putative antagonists, GM-109 and MA-2029 (modified 1-4 and 1-3 fragments). Activation and desensitization (2h preincubation with the motilin analogues 10muM) were studied in CHO-MTLR cells by an aequorin based luminescence assay. Phosphorylation was studied by immunoprecipitation and internalization was visualized in CHO-MTLR cells containing an enhanced green fluorescent protein (CHO-MTLR-EGFP).
Motilin [1-22] and [1-14] were more potent than Phe(3)[1-22] and Phe(3)[1-14] (pEC(50): 9.77, 8.78, 7.36 and 6.65, respectively) to induce Ca(2+) release. GM-109 and MA-2029 were without agonist activity. [1-22] and Phe(3)[1-22] decreased the second response to motilin from 78+/-2% to 11+/-3% and 34+/-3% (P<0.001), respectively, whereas [1-14], Phe(3)[1-14], GM-109 and MA-2029 had no desensitizing effect (68+/-5%, 78+/-3%, 78+/-6% and 78+/-5%, respectively, P>0.05). The rank order of MTLR-phosphorylation was: [1-22]>[1-14]>Phe(3)[1-22]=Phe(3)[1-14]>GM-109=MA-2029. Only motilin [1-22] and [1-14] induced receptor MTLR-EGFP internalization as shown by a decrease in membrane fluorescence: 20+/-3% and 7+/-3%, respectively.
The C-terminus of motilin enhances desensitization, phosphorylation and internalization of the MTLR while modifications of the N-terminus can favor a conformation of the receptor that is less susceptible to phosphorylation and internalization.
对胃肠肽胃动素片段的研究表明，该肽的C末端区域在胃动素受体（MTLR）脱敏中起重要作用。
为验证这一假设，我们研究了具有激动和拮抗特性的不同链长胃动素类似物在CHO-MTLR细胞中诱导的脱敏、磷酸化和内化。
我们研究了胃动素[1-22]、[1-14]片段、类似物Phe(3)[1-22]和Phe(3)[1-14]，以及两种假定的拮抗剂GM-109和MA-2029（修饰的1-4和1-3片段）。通过基于水母发光蛋白的发光测定法在CHO-MTLR细胞中研究激活和脱敏（用10μM胃动素类似物预孵育2小时）。通过免疫沉淀研究磷酸化，并在含有增强型绿色荧光蛋白的CHO-MTLR细胞（CHO-MTLR-EGFP）中观察内化。
胃动素[1-22]和[1-14]比Phe(3)[1-22]和Phe(3)[1-14]（pEC(50)分别为9.77、8.78、7.36和6.65）诱导Ca(2+)释放的能力更强。GM-109和MA-2029无激动剂活性。[1-22]和Phe(3)[1-22]分别将对胃动素的第二次反应从78±2%降至11±3%和34±3%（P<0.001），而[1-14]、Phe(3)[1-14]、GM-109和MA-2029无脱敏作用（分别为68±5%、78±3%、78±6%和78±5%，P>0.05）。MTLR磷酸化的顺序为：[1-22]>[1-14]>Phe(3)[1-22]=Phe(3)[1-14]>GM-109=MA-2029。只有胃动素[1-22]和[1-14]诱导受体MTLR-EGFP内化，表现为膜荧光降低：分别为20±3%和7±3%。
胃动素的C末端增强了MTLR的脱敏、磷酸化和内化，而N末端的修饰可使受体构象更不易发生磷酸化和内化。
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