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Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

Rizk SS, Kouadio JL, Szymborska A, Duguid EM, Mukherjee S, Zheng J, Clevenger CV, Kossiakoff AA - Cell Commun. Signal (2015)

Bottom Line: This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding.We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. srizk@nd.edu.

ABSTRACT

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.

Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.

Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

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Related in: MedlinePlus

sAB inhibition of hPRL-R in T47D breast cancer cells. a-b Fluorescence microscopy images of cells treated with 100 nM cy5-hPRL (a) or cy5-hGH (b) in the presence of each sAB. All panels represent two merged channels; blue: DAPI nuclear stain, red: cy5. c A Stat5 phospholyration luciferase reporter assay of T47D cells in the presence or absence of hPRL and hPRL-R sABs. A sAB against bacterial maltose binding protein (MBP) [7] was used as a negative control. d Western blot detection of the time-dependent inhibition of hPRL-R signaling by hPRL-R sABs.
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Fig2: sAB inhibition of hPRL-R in T47D breast cancer cells. a-b Fluorescence microscopy images of cells treated with 100 nM cy5-hPRL (a) or cy5-hGH (b) in the presence of each sAB. All panels represent two merged channels; blue: DAPI nuclear stain, red: cy5. c A Stat5 phospholyration luciferase reporter assay of T47D cells in the presence or absence of hPRL and hPRL-R sABs. A sAB against bacterial maltose binding protein (MBP) [7] was used as a negative control. d Western blot detection of the time-dependent inhibition of hPRL-R signaling by hPRL-R sABs.

Mentions: We note that biological signaling requires the hormones to bind the ECD at both Site1 and the lower affinity Site2. Thus, it is possible that the sABs have an even more profound effect on ECD binding at Site2. Unfortunately, because of its more transient nature, it is not possible to quantitatively measure the influence of sABs on binding of the hormone at Site2. Thus, we instead quantified the effects using relevant biological readouts of the intracellular signaling mechanism mediated by the hPRL-R using T47D, a breast cancer cell-line. First, we examined the effects of sABs on hormone-induced receptor internalization, which is a ubiquitous feature of cytokine receptor activation. We tested the ability of the four sABs to block hPRL-R-mediated internalization of cy5-labeled hPRL or hGH. Fluorescence microscopy indicated that sABs A8, A9 and A10 significantly decreased or completely abolished the internalization of hPRL, whereas the control sAB A4 had no effect on hormone internalization (Figure 2a). In contrast, internalization of hGH was only slightly inhibited by sABs A8, A9 and A10 (Figure 2b), presumably due to the ability of the sABs to recognize the conformation differences between the hPRL and hGH bound forms of hPRL-R.Figure 2


Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

Rizk SS, Kouadio JL, Szymborska A, Duguid EM, Mukherjee S, Zheng J, Clevenger CV, Kossiakoff AA - Cell Commun. Signal (2015)

sAB inhibition of hPRL-R in T47D breast cancer cells. a-b Fluorescence microscopy images of cells treated with 100 nM cy5-hPRL (a) or cy5-hGH (b) in the presence of each sAB. All panels represent two merged channels; blue: DAPI nuclear stain, red: cy5. c A Stat5 phospholyration luciferase reporter assay of T47D cells in the presence or absence of hPRL and hPRL-R sABs. A sAB against bacterial maltose binding protein (MBP) [7] was used as a negative control. d Western blot detection of the time-dependent inhibition of hPRL-R signaling by hPRL-R sABs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4300558&req=5

Fig2: sAB inhibition of hPRL-R in T47D breast cancer cells. a-b Fluorescence microscopy images of cells treated with 100 nM cy5-hPRL (a) or cy5-hGH (b) in the presence of each sAB. All panels represent two merged channels; blue: DAPI nuclear stain, red: cy5. c A Stat5 phospholyration luciferase reporter assay of T47D cells in the presence or absence of hPRL and hPRL-R sABs. A sAB against bacterial maltose binding protein (MBP) [7] was used as a negative control. d Western blot detection of the time-dependent inhibition of hPRL-R signaling by hPRL-R sABs.
Mentions: We note that biological signaling requires the hormones to bind the ECD at both Site1 and the lower affinity Site2. Thus, it is possible that the sABs have an even more profound effect on ECD binding at Site2. Unfortunately, because of its more transient nature, it is not possible to quantitatively measure the influence of sABs on binding of the hormone at Site2. Thus, we instead quantified the effects using relevant biological readouts of the intracellular signaling mechanism mediated by the hPRL-R using T47D, a breast cancer cell-line. First, we examined the effects of sABs on hormone-induced receptor internalization, which is a ubiquitous feature of cytokine receptor activation. We tested the ability of the four sABs to block hPRL-R-mediated internalization of cy5-labeled hPRL or hGH. Fluorescence microscopy indicated that sABs A8, A9 and A10 significantly decreased or completely abolished the internalization of hPRL, whereas the control sAB A4 had no effect on hormone internalization (Figure 2a). In contrast, internalization of hGH was only slightly inhibited by sABs A8, A9 and A10 (Figure 2b), presumably due to the ability of the sABs to recognize the conformation differences between the hPRL and hGH bound forms of hPRL-R.Figure 2

Bottom Line: This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding.We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. srizk@nd.edu.

ABSTRACT

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.

Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.

Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

Show MeSH
Related in: MedlinePlus