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Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

Bruell S, Kong RC, Petrie EJ, Hoare B, Wade JD, Scott DJ, Gooley PR, Bathgate RA - Front Endocrinol (Lausanne) (2013)

Bottom Line: However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal.Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy.Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

View Article: PubMed Central - PubMed

Affiliation: Florey Department of Neuroscience and Mental Health, Florey Institute of Neuroscience and Mental Health , Melbourne, VIC , Australia ; Department of Biochemistry and Molecular Biology , Melbourne, VIC , Australia.

ABSTRACT
Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

No MeSH data available.


Related in: MedlinePlus

Cell surface expression of chimeric receptors compared to the RXFP1 and RXFP2 wild-type (WT) receptors. Data are expressed as mean ± SEM of triplicate determinations from at least three independent experiments. **p < 0.01 compared to RXFP1.
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Figure 3: Cell surface expression of chimeric receptors compared to the RXFP1 and RXFP2 wild-type (WT) receptors. Data are expressed as mean ± SEM of triplicate determinations from at least three independent experiments. **p < 0.01 compared to RXFP1.

Mentions: Cell surface expression assays on the chimeric constructs showed that all receptors were expressed at the cell membrane, since their fluorescence profiles were consistently equal to or greater than that displayed by the WT receptors (Figure 3). Both RXFP211 and RXFP212 were expressed more highly than WT RXFP1, which is consistent with findings by Kern et al. for their LDLa-swapped chimera (12). However, only RXFP211 showed significantly (p < 0.01) higher expression than RXFP1 (141.2 ± 11.3% of RXFP1 expression) (Figure 3; Table 2).


Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

Bruell S, Kong RC, Petrie EJ, Hoare B, Wade JD, Scott DJ, Gooley PR, Bathgate RA - Front Endocrinol (Lausanne) (2013)

Cell surface expression of chimeric receptors compared to the RXFP1 and RXFP2 wild-type (WT) receptors. Data are expressed as mean ± SEM of triplicate determinations from at least three independent experiments. **p < 0.01 compared to RXFP1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3822782&req=5

Figure 3: Cell surface expression of chimeric receptors compared to the RXFP1 and RXFP2 wild-type (WT) receptors. Data are expressed as mean ± SEM of triplicate determinations from at least three independent experiments. **p < 0.01 compared to RXFP1.
Mentions: Cell surface expression assays on the chimeric constructs showed that all receptors were expressed at the cell membrane, since their fluorescence profiles were consistently equal to or greater than that displayed by the WT receptors (Figure 3). Both RXFP211 and RXFP212 were expressed more highly than WT RXFP1, which is consistent with findings by Kern et al. for their LDLa-swapped chimera (12). However, only RXFP211 showed significantly (p < 0.01) higher expression than RXFP1 (141.2 ± 11.3% of RXFP1 expression) (Figure 3; Table 2).

Bottom Line: However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal.Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy.Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

View Article: PubMed Central - PubMed

Affiliation: Florey Department of Neuroscience and Mental Health, Florey Institute of Neuroscience and Mental Health , Melbourne, VIC , Australia ; Department of Biochemistry and Molecular Biology , Melbourne, VIC , Australia.

ABSTRACT
Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

No MeSH data available.


Related in: MedlinePlus