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The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

Sethi A, Bruell S, Patil N, Hossain MA, Scott DJ, Petrie EJ, Bathgate RA, Gooley PR - Nat Commun (2016)

Bottom Line: H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1.Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation.We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry &Molecular Biology, The University of Melbourne, Victoria 3010, Australia.

ABSTRACT
H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

No MeSH data available.


H2 relaxin binding and activation of wild type and LDLa-linker mutants of RXFP1.(a) Saturation binding using Eu-H2 relaxin. (b) H2 relaxin-induced cAMP responses. (c) ML290-induced cAMP responses. Symbols represent mean values±s.e.m. from triplicate values in a minimum of three independent experiments.
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f2: H2 relaxin binding and activation of wild type and LDLa-linker mutants of RXFP1.(a) Saturation binding using Eu-H2 relaxin. (b) H2 relaxin-induced cAMP responses. (c) ML290-induced cAMP responses. Symbols represent mean values±s.e.m. from triplicate values in a minimum of three independent experiments.

Mentions: While the LDLa module of RXFP1 is indispensable for receptor activation10 and the LRR domain is considered as the primary H2 relaxin-binding site89, the role of the 32 residues linking the two domains has not been investigated. Here, we used transiently transfected human embryonic kidney (HEK) 293T cells expressing full-length RXFP1 and mutants to assess H2 relaxin binding and induced cAMP activation. Activation of these receptors was further tested with the small molecule RXFP1 agonist (ML290) that binds directly to the TMD activating RXFP1 in an allosteric and H2 relaxin-independent manner17. Initially we made three double mutants, G41A/D42A, N43A/N44A and G45A/W46A. These mutant receptors expressed at the cell surface and responded to ML290 activation comparable to wild-type receptor (Fig. 2c, Table 1) indicating that mutation of linker residues does not affect receptor trafficking or G-protein coupling. However, mutation of these residues had a profound effect on both H2 relaxin binding and activation. N43A/N44A demonstrated a fivefold loss of binding affinity for H2 relaxin, while G41A/D42A and G45A/W46A bound 25-fold more weakly (Fig. 2a, Table 1). Surprisingly the mutant receptors demonstrated reduced H2 relaxin potency, which was far greater than the reduction in affinity. N43A/N44A and G41A/D42A showed 500- and 5,000-fold loss of H2 relaxin-stimulated cAMP activity while G45A/W46A was virtually unable to signal (Fig. 2b, Table 1).


The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

Sethi A, Bruell S, Patil N, Hossain MA, Scott DJ, Petrie EJ, Bathgate RA, Gooley PR - Nat Commun (2016)

H2 relaxin binding and activation of wild type and LDLa-linker mutants of RXFP1.(a) Saturation binding using Eu-H2 relaxin. (b) H2 relaxin-induced cAMP responses. (c) ML290-induced cAMP responses. Symbols represent mean values±s.e.m. from triplicate values in a minimum of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837482&req=5

f2: H2 relaxin binding and activation of wild type and LDLa-linker mutants of RXFP1.(a) Saturation binding using Eu-H2 relaxin. (b) H2 relaxin-induced cAMP responses. (c) ML290-induced cAMP responses. Symbols represent mean values±s.e.m. from triplicate values in a minimum of three independent experiments.
Mentions: While the LDLa module of RXFP1 is indispensable for receptor activation10 and the LRR domain is considered as the primary H2 relaxin-binding site89, the role of the 32 residues linking the two domains has not been investigated. Here, we used transiently transfected human embryonic kidney (HEK) 293T cells expressing full-length RXFP1 and mutants to assess H2 relaxin binding and induced cAMP activation. Activation of these receptors was further tested with the small molecule RXFP1 agonist (ML290) that binds directly to the TMD activating RXFP1 in an allosteric and H2 relaxin-independent manner17. Initially we made three double mutants, G41A/D42A, N43A/N44A and G45A/W46A. These mutant receptors expressed at the cell surface and responded to ML290 activation comparable to wild-type receptor (Fig. 2c, Table 1) indicating that mutation of linker residues does not affect receptor trafficking or G-protein coupling. However, mutation of these residues had a profound effect on both H2 relaxin binding and activation. N43A/N44A demonstrated a fivefold loss of binding affinity for H2 relaxin, while G41A/D42A and G45A/W46A bound 25-fold more weakly (Fig. 2a, Table 1). Surprisingly the mutant receptors demonstrated reduced H2 relaxin potency, which was far greater than the reduction in affinity. N43A/N44A and G41A/D42A showed 500- and 5,000-fold loss of H2 relaxin-stimulated cAMP activity while G45A/W46A was virtually unable to signal (Fig. 2b, Table 1).

Bottom Line: H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1.Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation.We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry &Molecular Biology, The University of Melbourne, Victoria 3010, Australia.

ABSTRACT
H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

No MeSH data available.