Low-density Lipoprotein Class A Research Articles

In a Class of Their Own - RXFP1 and RXFP2 are Unique Members of the LGR Family.

The leucine-rich repeat-containing G protein-coupled receptors (LGRs) family consists of three groups: types A, B, and C and all contain a large extracellular domain (ECD) made up of the structural motif – the leucine-rich repeat (LRR). In the LGRs, the ECD binds the hormone or ligand, usually through the LRRs, that ultimately results in activation and signaling. Structures are available for the ECD of type A and B LGRs, but not the type C LGRs. This review discusses the structural features of LRR proteins, and describes the known structures of the type A and B LGRs and predictions that can be made for the type C LGRs. The mechanism of activation of the LGRs is discussed with a focus on the role of the low-density lipoprotein class A (LDLa) module, a unique feature of the type C LGRs. While the LDLa module is essential for activation of the type C LGRs, the molecular mechanism for this process is unknown. Experimental data for the potential interactions of the type C LGR ligands with the LRR domain, the transmembrane domain, and the LDLa module are summarized.

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.

Mapping key regions of the RXFP2 low-density lipoprotein class-A module that are involved in signal activation.

The peptide hormone INSL3 and its receptor, RXFP2, have co-evolved alongside relaxin and its receptor, RXFP1. Both RXFP1 and RXFP2 are G protein-coupled receptors (GPCRs) containing the hallmark seven transmembrane helices in addition to a distinct ectodomain of leucine-rich repeats (LRRs) and a single low-density lipoprotein class-A (LDLa) module at the N-terminus. RXFP1 and RXFP2 are the only mammalian GPCRs known to contain an LDLa, and its removal does not perturb primary ligand binding to the LRRs; however, signaling is abolished. This presents a general mechanism whereby ligand binding induces a conformational change in the receptor to position the LDLa to elicit a signal response. Although the LDLa interaction site has not been identified, the residues important to the action have been mapped within the RXFP1 LDLa module. In this study, we comprehensively study the RXFP2 LDLa module. We determine its structure using nuclear magnetic resonance (NMR) and concurrently investigate the signaling of an RXFP2 with the LDLa removed (RXFP2-short), confirming that the LDLa is essential to signaling. We then replaced the LDLa with the second ligand binding module from the LDL receptor, LB2, creating the RXFP2-LB2 chimera. Unlike that in the equivalent RXFP1-LB2 chimera, signaling is rescued albeit modestly. Guided by the NMR structure, we dissected regions of the RXFP2 LDLa to identify specific residues that are important to signal activation. We determine that although the module is important to the activation of RXFP2, unlike the RXFP1 receptor, specific residues in the N-terminus of the domain are not involved in signal activation.

The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

Diversity of lectins in Macrobrachium rosenbergii and their expression patterns under spiroplasma MR-1008 stimulation

Lectins play important roles in crustacean innate immunity through recognition of foreign pathogens. In this study, 20 lectins including C-type lectins [dual-carbohydrate recognition domain (CRD) type and single-CRD type], L-type lectin, and lectin with low-density lipoprotein class A (LDLa) domain were identified from the freshwater prawn Macrobrachium rosenbergii. The tissue distribution and expression patterns of these lectins under spiroplasma strain MR-1008 challenge were investigated. Most of the lectins were found to be mainly distributed in the hepatopancreas. Lectin5, Lectin14, Lectin17, and Lectin18 exhibited the highest expression level in the hemocytes, nerve, intestine, and heart, respectively. MrLec1 to MrLec6 (dual-CRD lectins) in the hepatopancreas were up-regulated by spiroplasma challenge. Single-CRD lectins reached the highest level at 72 h after spiroplasma challenge. Lectin9 and Lectin15 both belong to L-type lectins. At post-spiroplasma challenge, Lectin9 expression was up-regulated, whereas Lectin15 expression was down-regulated. Lectin11 with LDLa domain showed the highest level after 12 h Lectin18 and Lectin20, namely, CD209, were also up-regulated by spiroplasma challenge. Lectin14, a C-type lectin, quickly reached the highest level after 2 h Lectin16 showed the highest level after 72 h Lectin5 reached the highest level in cultured hemocytes after 6 h Lectin17 in the intestine and Lectin14 in the nerve were slightly up-regulated after 6 and 2 h, respectively. Our research results indicate that lectins may play important roles in early or late immune responses against spiroplasma challenge.

A novel protein engineering approach for investigating GPCR/ligand interactions

Relaxin family peptide receptors (RXFP) 1 and 2, receptors for relaxin and insulin like peptide‐3 (INSL3) respectively, are G‐protein coupled receptors possessing large N terminal domains consisting of 10 leucine rich repeats (LRRs) and a low density lipoprotein Class A (LDLa) module. Ligand mediated activation requires primary binding to the LRRs as well as a secondary interaction involving the extracellular loops (ELs) of the transmembrane domains. The LDLa is necessary for activation possibly by a specific interaction with the ELs.The project aims to identify residues of RXFP1 and RXFP2 ELs involved in ligand/LDLa binding by mounting them onto a soluble protein scaffold and mapping specific ligand interactions using NMR. We have produced soluble scaffolds containing RXFP1 and RXFP2 EL1 and EL2 and are conducting ligand/LDLa titrations for NMR analysis before residue assignment. Preliminary NMR results show peak changes upon relaxin titration with the RXFP1 scaffold suggesting specific interactions are occurring. Identified interacting residues will be mutated in RXFP1 and functional characterisation will confirm their importance for receptor function.Understanding mechanisms of RXFP1 and RXFP2 activation will aid design of relaxin and INSL3 analogues for therapeutic use. This work is supported by project grant 628427 (to RADB and PRG) from the National Health and Medical Research Council, Australia.

Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function

The receptors for relaxin and insulin-like peptide 3 (INSL3) are now well-characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G-protein-coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine-rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low-density lipoprotein class A (LDL-A) module at their N-terminus. Ligand-mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL-A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL-A module is currently unknown. The specific understanding of the mechanisms underlying ligand-mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.

Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function

The LGR7/RXFP1 and LGR8/RXFP2 receptors are unique receptors among the G-protein-coupled receptors (GPCRs) in having a low-density lipoprotein class A (LDL-A) module. Their complex gene organization, among the intron-richest of the GPCRs, suggests that alternative splicing is a common occurrence. We have therefore investigated the role of the LDL-A module and shown the identity, expression, and functions of three LGR7 splice variants in the human decidua. Point mutations of conserved residues or complete deletion of the LDL-A module resulted in loss of the cAMP response to relaxin. Its glycosylation also impacted LGR7 cell surface delivery and therefore receptor activation. The wild-type (WT) LGR7 was expressed as both precursor and mature forms, but deletion of the LDL-A module resulted in expression of only the mature form. Three new alternatively spliced variants of LGR7 were identified, all containing a truncated extracellular region. Their functional characterization showed them exerting dominant negative effects on the WT LGR7 by preventing its homodimerization, maturation, and subsequent trafficking to the cell surface, resulting in loss of function. In summary, different mechanisms have been identified for controlling the cell surface expression and function of the LGR7 protein which are likely to be significant for the role of relaxin in human parturition.

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

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.

The Long and the Short of Relaxin Signaling

The hormone relaxin functions in the reproductive system and derived its name from its role in relaxation of the pubic symphysis during parturition. Relaxin and the related hormone insulin-like peptide 3 (INSL3) act through heterotrimeric guanine nucleotide­-binding protein (G protein)-coupled receptors (GPCRs). Scott et al . report studies of human splice variants of these proteins that reveal an unusual mode of signaling by these receptors. The receptors for relaxin and INSL3, called LGR7 (leucine-rich repeat-containing GPCR 7) and LGR8, respectively [or, in an alternative nomenclature, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2)] differ from other GPCRs in that they contain N-terminal domains with similarity to the ligand-binding domains of low-density lipoprotein class A (LDLa). Splice variants exist in which this LDLa module is missing or in which a secreted protein mainly encoding just the LDLa module is made. The authors examined the signaling properties of natural or artificial variant receptors in transfected cells. Although they bound relaxin normally, LGR7 variants without the LDLa module failed to produce their normal G protein-coupled signaling effects on accumulation of cyclic AMP. The truncated excreted form inhibited signaling from the normal receptor. It is not yet clear precisely how the LDLa modules influence signaling. In LDL receptors, these modules are binding sites for ligands or for protein-protein interactions. The truncated, excreted form of LDL7 was detected in human uterus and brain and was most abundant in uteri of pregnant mice. Thus, the authors propose that LGR7 and LGR8 have an unusual mechanism of GPCR signaling and that the alternative splicing of the receptors may produce an inhibitory factor that provides an additional regulatory mechanism for controlling relaxin action during reproduction. D. J. Scott, S. Layfield, Y. Yan, S. Sudo, A. J. W. Hsueh, G. W. Tregear, R. A. D. Bathgate, Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J. Biol. Chem. 281 , 34942-34954 (2006). [Abstract] [Full Text]

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

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.

The Human LGR7 Low‐Density Lipoprotein Class A Module Requires Calcium for Structure

The relaxin and INSL3 receptors, LGR7 and LGR8, are the only human G-protein-coupled receptors to contain a low-density lipoprotein class-A (LDL-A) module. LDL-A modules are well characterized in a variety of diverse biological functions that involve ligand binding to elicit a response. Common features of the LDL-A modules characterized to date are the conservation of three disulfide bonds and the structural arrangement around a bound calcium ion. In this study we recombinantly produce the human LGR7 LDL-A module for NMR studies and demonstrate that calicum is required for the module to form a stable and correctly folded structure.

A novel gene encoding a putative transmembrane protein with two extracellular CUB domains and a low-density lipoprotein class A module: isolation of alternatively spliced isoforms in retina and brain

We report herein the cDNA cloning of a novel retina and brain specific gene from mouse and human encoding a putative transmembrane protein with an N-terminal signal sequence and two conserved extracellular CUB domains followed by a single copy of the low-density lipoprotein class A (LDLa) module. The mouse and human genes, termed NETO1 (neuropilin and tolloid like-1), display sequence identities of 87% at the nucleotide and 95% at the protein level. The human NETO1 gene comprises 13 exons on chromosome 18q22–q23 and gives rise to three different mRNA isoforms. Two alternative leader exons 1a and 1b generate transcripts that translate into putative signal peptides with individual sequence composition but otherwise do not affect the primary structure of the mature NETO1 protein. Usage of the internal exon 5 is restricted to the retinal tissue and generates a truncated transcript that codes for a putative soluble protein, termed sNETO1, with only one copy of the CUB domain while lacking the LDLa module. NETO1 exhibits 57% identity to the deduced amino acid sequence of a non-annotated nucleotide sequence in the GenBank database, therefore designated NETO2. Both NETO1 and NETO2 share an identical and unique domain structure thus representing a novel subfamily of CUB- and LDLa-containing proteins. The cytoplasmic domains of NETO1 and NETO2 are not homologous to other known protein sequences but contain a conserved FXNPXY-like motif, which is essential for the internalization of clathrin coated pits during endocytosis or alternatively, may be implicated in intracellular signaling pathways.