Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors

Ahmed, Shawn; Murphy, Richard F.; Lovas, Sándor

doi:10.1016/j.peptides.2005.02.001

Cited by 10 publications

(9 citation statements)

References 17 publications

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“…Growth factor effects of PG and G-Gly peptides are mediated by novel receptor mechanisms, distinct from CCK 2 R and CCK 1 R (Seva et al, 1994;Ahmed et al, 2005;Rengifo-Cam et al, 2007). Earlier, we had identified a 33-36 kDa protein as a high-affinity binding protein for PG/gastrin peptides, which was pharmacologically different from CCK 1 R and CCK 2 R (Chicone et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

et al. 2008

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Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IjBa (IKKa/b), inhibitor of jB (IjB)a and p65NF-jB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IjBa and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly colocalized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-jB (NF-jB) activation), demonstrating for the first time a critical role of NF-jB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-jB may significantly reduce the increased risk of colon carcinogenesis in response to PG.

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Section: Discussionmentioning

confidence: 99%

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

et al. 2008

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“…The low binding might be either to the G17-Gly low affinity site, or to the G17-Gly high affinity site. [Leu 15 ]G17(6–17)-Gly and [Leu 15 ]G17(11–17)-Gly also bound one site with similarly low affinity and displacement of receptors [2]. …”

Section: Discussionmentioning

confidence: 99%

“…Peptides were analyzed and purified on a dual pump HPLC apparatus (Gilson, Middleton, WI, USA) as previously described [2, 11]. For analytical HPLC either a Vydac 218TP54 column (C 18 , 5 μm particle size, 4.6 mm × 250 mm) (Grace Vydac, Hesperia, CA, USA) or a Phenomenex 00G-4252-Y0 “Luna” column (C 18 , 5 μm particle size, 3 mm × 250 mm) (Phenomenex, Torrance, CA, USA) were used at flow rates of 1 mL/min and 0.5 mL/min, respectively, with an initial injection concentration of 1 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently it was shown that these two sites are responsible for a biphasic growth effect when DLD-1 cells are treated with G17-Gly. Further studies revealed that both C-terminal analogs [Leu 15 ]G17(6–17)-Gly and [Leu 15 ]G17(11–17)-Gly bind to a single site on DLD-1 cells with close to micromolar affinity, while the N-terminal analog G17(1–12) stimulates nonbiphasic proliferation of HT-29 cells [2]. These results suggest that the N-terminal region of G17 is essential for binding and activation of a nanomolar affinity receptor that mediates the growth-promoting effects of the peptide.…”

Section: Introductionmentioning

confidence: 99%

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Bioactivity of analogs of the N-terminal region of gastrin-17

et al. 2009

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Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1–12) and then to G17(1–6)-NH2 did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1–4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1–12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1–6)-NH2, including nonamidated G17(1–6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1–6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.

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“…In previous work, this group confirmed the existence of nanomolar and micromolar affinity receptors on the DLD-1 and HT-29 colon cancer cell lines [2]. It was also demonstrated that the N-terminal region of G17, G17(1-12), was able to stimulate the proliferation of HT-29 cells, showing that the C-terminal tetrapeptide (Trp-Met-Asp-Phe-NH 2 ) of G17 which is essential for binding and activation of the CCK2 receptor is not necessary for binding and activation of the putative G17-Gly growth promoting receptor [3]. Furthermore, we have shown [13] that 1, G17(1-12) binds to high and low affinity receptors on DLD-1 cells as does [Leu 15 ]G17-Gly and promotes the proliferation of such cells; 2, further excision of the sequence Glu 7 -Tyr 12 still gives a peptide, G17(1-6)-NH 2 , which binds, albeit with low affinity, and activates the putative growth promoting receptor on DLD-1 cells; 3, shorter N-terminal analogs with and without C-terminal amidation, including G17(1-6), bind with decreasing affinity than that of G17(1-6)-NH 2 [15].…”

Section: Introductionmentioning

confidence: 99%

The structure of bioactive analogs of the N-terminal region of gastrin-17

2009

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Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1-12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1-4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1-6)-NH 2 and G17(1-12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange Molecular Dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a β-turn at residues 1-4, were found in most peptides by REMD simulations. G17(1-3)-NH 2 , which cannot form a β-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the β-turn appeared more frequently longer sequences, perhaps explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1-6)-NH 2 to activate the non-CCK receptor where as the non-amidated G17(1-6) and shorter peptides do not.

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Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors

Cited by 10 publications

References 17 publications

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Bioactivity of analogs of the N-terminal region of gastrin-17

The structure of bioactive analogs of the N-terminal region of gastrin-17

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