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 receptors for the peptide hormones relaxin and insulin-like peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa module of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity.     

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 A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.     

H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.     

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.     

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.     

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors.

Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.     

Defining the LGR8 residues involved in binding insulin-like peptide 3

The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [(125)I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.     

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4–24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.     

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.     

The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist

Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist; however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.     

Role of asparagine-linked oligosaccharides in the function of the rat PTH/PTHrP receptor

The receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP) is a G-protein-coupled receptor with four potential sites for N-linked glycosylation. The contribution of the oligosaccharide moieties to cell surface expression, ligand binding, and signal transduction was investigated. Site-directed mutagenesis of the rat PTH/PTHrP receptor cDNA was performed at single or combination of the four potential glycosylation sites to determine the effect of the putative carbohydrate chains on the activities of the receptor. The results revealed that all four potential N-glycosylation sites in the PTH/PTHrP receptor are glycosylated. Receptors missing a single or multiple glycosylation consensus but with at least one intact glycosylation site expressed sufficiently and functioned normally. In contrast, the nonglycosylated receptor, in which all four glycosylation sites were mutated, is deficient in these functions. These data indicate important roles for N-linked glycosylation in PTH/PTHrP receptor functions.     

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.     

The trap-like relaxin-binding site of the leucine-rich G-protein-coupled receptor 7

The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of approximately 45 degrees across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain alpha-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20.     

The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.     

Proteolytic activation and glycosylation of N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme involved in the endocannabinoid metabolism

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme hydrolyzing bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. Previously, we suggested that NAAA is glycosylated and proteolytically cleaved. Here, we investigated the mechanism and significance of the cleavage of human NAAA overexpressed in human embryonic kidney 293 cells. Western blotting with anti-NAAA antibody revealed that most of NAAA in the cell homogenate was the cleaved 30-kDa form. However, some of NAAA were released outside the cells and the extracellular enzyme was mostly the uncleaved 48-kDa form. When incubated at pH 4.5, the 48-kDa form was time-dependently converted to the 30-kDa form with concomitant increase in the N-palmitoylethanolamine-hydrolyzing activity. The purified 48-kDa form was also cleaved and activated. However, the cleavage did not proceed at pH 7.4 or in the presence of p-chloromercuribenzoic acid. The mutant C126S was resistant to the cleavage and remained inactive. These results suggested that this specific proteolysis is a self-catalyzed activation step. We next determined N-glycosylation sites of human NAAA by site-directed mutagenesis addressed to asparagine residues in six potential N-glycosylation sites. The results exhibited that Asn-37, Asn-107, Asn-309, and Asn-333 are actual N-glycosylation sites. The glycosylation appeared to play an important role in stabilizing the enzyme protein.     

N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling

To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.