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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

Schematic representation of the RXFP1 and RXFP2 receptors compared to the RXFP211, RXFP122, RXFP212, and RXFP121 chimeric receptors. The domains of receptors are labeled on the RXFP1 receptor. RXFP1 domains are in blue while RXFP2 domains are in red.
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Figure 2: Schematic representation of the RXFP1 and RXFP2 receptors compared to the RXFP211, RXFP122, RXFP212, and RXFP121 chimeric receptors. The domains of receptors are labeled on the RXFP1 receptor. RXFP1 domains are in blue while RXFP2 domains are in red.

Mentions: The present study aims to further investigate this seeming contradiction, and establish why an ectodomain-swapped chimera of RXFP1 with RXFP2 can signal, while an equivalent LDLa-swapped chimera cannot. Upon scrutiny of the constructs made by Kern et al., it would appear that the point chosen to swap the LDLa modules was 27 residues C-terminally to the final cysteine (Cys40) necessary for LDLa structural integrity for RXFP1. Importantly, the equivalent region of RXFP2 which was replaced in the chimera is seven amino acids shorter (Figure 1), meaning that the resulting chimera had a shorter stretch of amino acids linking the LDLa module to the LRRs than WT RXFP1. We therefore designed chimeric constructs of both RXFP1 and RXFP2 that had their LDLa modules swapped immediately C-terminally to the aforementioned cysteine similar to our RXFP1-LB2 chimera (8), meaning that only the LDLa module itself was swapped, and none of the adjoining linker residues. We call these constructs RXFP211 and RXFP122 (Figure 2) based on the nomenclature utilized previously for ectodomain-swapped RXFP1 and RXFP2 receptors (11). As further investigation into the possibility of a receptor-specific interaction between the LDLa module and the TM domain, we also matched the LDLa module with the complementary TM domain, to establish if an improvement in signaling activity would be observed. These constructs are named RXFP212 and RXFP121 (Figure 2). The resulting chimeras were tested for surface expression, binding of ligand, and cAMP-based signaling.


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)

Schematic representation of the RXFP1 and RXFP2 receptors compared to the RXFP211, RXFP122, RXFP212, and RXFP121 chimeric receptors. The domains of receptors are labeled on the RXFP1 receptor. RXFP1 domains are in blue while RXFP2 domains are in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Schematic representation of the RXFP1 and RXFP2 receptors compared to the RXFP211, RXFP122, RXFP212, and RXFP121 chimeric receptors. The domains of receptors are labeled on the RXFP1 receptor. RXFP1 domains are in blue while RXFP2 domains are in red.
Mentions: The present study aims to further investigate this seeming contradiction, and establish why an ectodomain-swapped chimera of RXFP1 with RXFP2 can signal, while an equivalent LDLa-swapped chimera cannot. Upon scrutiny of the constructs made by Kern et al., it would appear that the point chosen to swap the LDLa modules was 27 residues C-terminally to the final cysteine (Cys40) necessary for LDLa structural integrity for RXFP1. Importantly, the equivalent region of RXFP2 which was replaced in the chimera is seven amino acids shorter (Figure 1), meaning that the resulting chimera had a shorter stretch of amino acids linking the LDLa module to the LRRs than WT RXFP1. We therefore designed chimeric constructs of both RXFP1 and RXFP2 that had their LDLa modules swapped immediately C-terminally to the aforementioned cysteine similar to our RXFP1-LB2 chimera (8), meaning that only the LDLa module itself was swapped, and none of the adjoining linker residues. We call these constructs RXFP211 and RXFP122 (Figure 2) based on the nomenclature utilized previously for ectodomain-swapped RXFP1 and RXFP2 receptors (11). As further investigation into the possibility of a receptor-specific interaction between the LDLa module and the TM domain, we also matched the LDLa module with the complementary TM domain, to establish if an improvement in signaling activity would be observed. These constructs are named RXFP212 and RXFP121 (Figure 2). The resulting chimeras were tested for surface expression, binding of ligand, and cAMP-based signaling.

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