Characterization of Novel Splice Variants of LGR7 and LGR8 Reveals That Receptor Signaling Is Mediated by Their Unique Low Density Lipoprotein Class A Modules

Abstract

The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.Relaxin was initially named for its ability to relax the pubic symphysis in pregnant guinea pigs at parturition (1). Since then relaxin has been found to be involved in many physiological processes, including cervical ripening (2-5), inhibition of myometrial contractions in some mammals (6 -8), uterine growth during pregnancy (9, 10), and nipple development for lactation (11-13). Most of the actions of relaxin are a direct result of its ability to stimulate the breakdown and remodeling of collagen fibers by inhibiting collagen type I and III synthesis and promoting matrix metalloproteinase expression and activation (14 -16). Most mammalian species have relaxin; however, due to a gene duplication event, humans possess two relaxin genes, encoding H1 relaxin and H2 relaxin, with H2 relaxin being the major stored and circulating form (reviewed in Ref. 17). In pig, mouse, rat, and human, the primary source of relaxin is the corpus luteum (reviewed in Ref. 18), highlighting that the most pronounced roles of relaxin occur during pregnancy.The relaxin receptor is a GPCR 3 most recently named the RXFP1 receptor (relaxin family peptide receptor 1) (19), however, in this report it will be referred to by its original name, leucine-rich repeat-containing GPCR 7 (LGR7) (20). LGR7 has been highly conserved in vertebrate species throughout evolution and is related...

The NMR Solution Structure of the Relaxin (RXFP1) Receptor Lipoprotein Receptor Class A Module and Identification of Key Residues in the N-terminal Region of the Module That Mediate Receptor Activation

“…The orientation of the side chains of residues Leu-7, Tyr-9, Leu-22, and Leu-23 suggest these residues cluster to create a hydrophobic surface on the molecule. N-Linked glycosylation of Asn-14 has been reported recently not to be essential for receptor activity (28), suggesting that this region is not involved in the primary activation of RXFP1 receptor signaling. (Table 2), which is much higher than the K d of 20 M for wild type protein.…”

“…The LDLa module of RXFP1 is essential for receptor activation (28). To demonstrate that the function of the RXFP1 LDLa module depends on specific side chain interactions, we replaced it with the second ligand binding domain (LB2) of the LDL receptor producing the chimera LB2-RXFP1.…”

“…The pcDNA3.1/Zeo plasmid containing the LGR7 short construct (28), missing the LDLa module, retained an EcoRI site following the bovine prolactin signal sequence and FLAG epitope, prior to the beginning of the LGR7 sequence. The LB2 insert and the pcDNA3.1 LGR7 short plasmid were therefore simultaneously digested with EcoRI and ligated together to create the LB2-RXFP1 chimeric receptor.…”

“…There is no evidence to suggest that the LDLa module is involved in the binding of relaxin. Conversely, the LDLa module has been demonstrated to be essential to the activation of signaling of the RXFP1 receptor (28). Removal of the LDLa module has no effect on the affinity of relaxin binding but abolishes the capacity of the truncated receptor to activate the response of cAMP production.…”

Alternative splicing of G protein-coupled receptors: physiology and pathophysiology

“…Several splice variants of RXFP1 (formerly known as LGR7 (leucine-rich-repeat-containing G protein-coupled receptor 7)) have been identified. Some of them produce a soluble, truncated and secreted protein which binds to relaxin and antagonizes its actions in vivo [73]. Several splice variants, cloned from human foetal membranes [74,75], human uterus and brain [130], encode RXFP1 isoforms with different lengths of the extracellular domain.…”