The Low-Density Lipoprotein Class A Module of the Relaxin Receptor (Leucine-Rich Repeat Containing G-Protein Coupled Receptor 7): Its Role in Signaling and Trafficking to the Cell Membrane

Abstract

The relaxin receptor (LGR7, relaxin family peptide receptor 1) is a member of the leucine-rich repeat containing G protein-coupled receptors subgroup C. This and the LGR8 (relaxin family peptide receptor 2) receptor are unique in having a low-density lipoprotein class A (LDL-A) module at their N termini. This study was designed to show the role of the LDL-A in LGR7 expression and function. Point mutants for the conserved cysteines (Cys(47) and Cys(53)) and for calcium binding asparagine (Asp(58)), a mutant with deleted LDL-A domain and chimeric LGR7 receptor with LGR8 LDL-A all showed no cAMP response to human relaxins H1 or H2. We have shown that their cell surface delivery was uncompromised. The mutation of the putative N-linked glycosylation site (Asn(36)) decreased cAMP production and reduced cell surface expression to 37% of the wild-type LGR7. All point mutant, chimeric, and wild-type receptor proteins were expressed as the two forms. The immature or precursor form of the receptor was 80 kDa, whereas the mature receptor, delivered to the cell surface was 95 kDa. The glycosylation mutant was also expressed as two forms with appropriately smaller molecular masses. Deletion of the LDL-A module resulted in expression of the mature receptor only. These data suggest that the LDL-A module of LGR7 influences receptor maturation, cell surface expression, and relaxin-activated signal transduction.

Evidence for expression of relaxin hormone-receptor system in the boar testis

“…In the present study, the protein encoded by RXFP1 was translated into three protein species with apparent molecular masses of 75, 91 and 95 kDa in the testis during development. Taken with previous findings (Kern et al 2007), specific bands of both 91 and 95 kDa detected in the boar testis are likely to be glycosylated forms of the RXFP1 receptor, whereas the 75 kDa band may represent a non-glycosylated form, although it is somewhat smaller in size than the non-glycosylated human RXFP1.…”

“…Members of this family of receptors have N-glycosylation sites in the ectodomains and act through causing cAMP production (Hsu et al 2002, Yan et al 2008b. Expression analysis and glycosidase treatment have shown that human RXFP1 is expressed as two molecular masses of 80 and 95 kDa in human embryonic kidney cells (HEK293 cells) transfected with the full-length RXFP1, and that while the 95 kDa band represents the glycosylated form of the RXFP1 receptor, the 80 kDa band represents the non-glycosylated form (Kern et al 2007). In the present study, the protein encoded by RXFP1 was translated into three protein species with apparent molecular masses of 75, 91 and 95 kDa in the testis during development.…”

Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment

“…6C). A previous study by Kern et al (25) involving immunoprecipitation and Western blotting for HA-tagged RXFP1 shows the presence of two distinct immunoprecipitated bands, at 95 kDa and 80 kDa, the larger of which corresponds to the mature form of the receptor (25). In our hands, FLAG-tagged RXFP1 migrates as a broad single band at 80 -90 kDa, and the mature and immature forms are unable to be distinguished (52).…”

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…In addition to the LRR region, the N-terminal LDLa module is essential for signaling ( Fig. 1) but has no role in ligand binding Hopkins et al, 2007;Kern et al, 2007). The LDLa module likely interacts with other receptor domains (potentially the ECLs and TM domains) in a manner analogous to a tethered ligand (Fig.…”