Large Ectodomain Research Articles

Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor.

Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor (HGF/SF). Here, we establish by deletion mutagenesis that the HGF/SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the alpha-chain (amino acids 25-307) and the first 212 amino acids of the beta-chain (amino acids 308-519). Neither the cystine-rich domain (amino acids 520-561) nor the C-terminal half of MET (amino acids 562-932) bind HGF/SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF/SF in the absence or presence of heparin, and forms a stable HGF/SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a beta-propeller fold, which is similar to the N-terminal domain of alphaV integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839-924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structure-function of the MET receptor and the related semaphorin and plexin proteins.

Tamm-Horsfall glycoprotein: biology and clinical relevance

Tamm-Horsfall glycoprotein (THP) is the most abundant urinary protein in mammals. Urinary excretion occurs by proteolytic cleavage of the large ectodomain of the glycosyl phosphatidylinositol-anchored counterpart exposed at the luminal cell surface of the thick ascending limb of Henle's loop. We describe the physical-chemical structure of human THP and its biosynthesis and interaction with other proteins and leukocytes. The clinical relevance of THP reported here includes: (1) involvement in the pathogenesis of cast nephropathy, urolithiasis, and tubulointerstitial nephritis; (2) abnormalities in urinary excretion in renal diseases; and (3) the recent finding that familial juvenile hyperuricemic nephropathy and autosomal dominant medullary cystic kidney disease 2 arise from mutations of the THP gene. We critically examine the literature on the physiological role and mechanism(s) that promote urinary excretion of THP. Some lines of research deal with the in vitro immunoregulatory activity of THP, termed uromodulin when isolated from urine of pregnant women. However, an immunoregulatory function in vivo has not yet been established. In the most recent literature, there is renewed interest in the capacity of urinary THP to compete efficiently with urothelial cell receptors, such as uroplakins, in adhering to type 1 fimbriated Escherichia coli. This property supports the notion that abundant THP excretion in urine is promoted in the host by selective pressure to obtain an efficient defense against urinary tract infections caused by uropathogenic bacteria.

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit.

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.

Topology of hepatitis C virus envelope glycoproteins.

Hepatitis C virus encodes two envelope glycoproteins, E1 and E2, that are released from a polyprotein precursor after cleavage by host signal peptidase(s). These proteins contain a large N-terminal ectodomain and a C-terminal transmembrane domain, and they assemble as a noncovalent heterodimer. The transmembrane domains of hepatitis C virus envelope glycoproteins have been shown to be multifunctional: (1) they are membrane anchors, (2) they bear ER retention signals, (3) they contain a signal sequence function, and (4) they are involved in E1-E2 heterodimerisation. Due to these multiple functions, the topology adopted by these transmembrane domains has given rise to much controversy. They are less than 30 amino acid residues long and are composed of two stretches of hydrophobic residues separated by a short segment containing one or two fully conserved positively charged residues. The presence of a signal sequence function in the C-terminal half of the transmembrane domains of E1 and E2 had suggested that these domains are composed of two membrane spanning segments. However, the two hydrophobic stretches are too short to make two membrane spanning alpha-helices. These discrepancies can now be explained by a dynamic model, based on experimental data, describing the early steps of the biogenesis of hepatitis C virus envelope glycoproteins. In this model, the transmembrane domains of E1 and E2 form a hairpin structure before cleavage by a signal peptidase, and a reorientation of the second hydrophobic stretch occurs after cleavage to produce a single membrane spanning domain.

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.

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling.

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.

Structural Requirements for Mutational Lutropin/Choriogonadotropin Receptor Activation

Different activation mechanisms of glycoprotein hormone receptors, which are members of the G protein-coupled receptor superfamily, have been proposed. For example, the large ectodomain of glycoprotein hormone receptors may function as an inverse agonist keeping the transmembrane domain in an inactive conformation. To provide support for this hypothesis, we have generated different lutropin/choriogonadotropin receptor (LHR) constructs lacking the ectodomain. Although some ectodomain-deficient LHR constructs were targeted to the cell surface, cAMP levels remained unchanged under basal conditions and agonist application but could be increased by a mutation within the transmembrane domain 6 (D578H). Taking advantage of a constitutive activating mutation (S277N) located in the extracellular domain, we showed that the intact leucine-rich repeat-containing ectodomain is essential for constitutive activation of the LHR by mutation of the hinge region. Our findings support an activation scenario in which agonist binding or mutational alterations expose a structure within the ectodomain, which then activates the transmembrane core.

Three-dimensional model of the human platelet integrin alpha IIbbeta 3 based on electron cryomicroscopy and x-ray crystallography.

Integrins are a large family of heterodimeric transmembrane signaling proteins that affect diverse biological processes such as development, angiogenesis, wound healing, neoplastic transformation, and thrombosis. We report here the three-dimensional structure at 20-A resolution of the unliganded, low-affinity state of the human platelet integrin alpha(IIb)beta(3) derived by electron cryomicroscopy and single particle image reconstruction. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as two parallel transmembrane alpha-helices. The docking of the x-ray structure of the alpha(V)beta(3) ectodomain into the electron cryomicroscopy map of alpha(IIb)beta(3) requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high- and low-affinity conformations reveals dramatic conformational changes associated with integrin activation.

Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes.

Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane alpha helices and small ecto- and endodomains. A His6-tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-beta-d-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.

Human secreted attractin disrupts neurite formation in differentiating cortical neural cells in vitro.

Mutations at the Atrn locus that encodes a transmembrane protein with a large ectodomain are responsible for a juvenile-onset neurodegeneration manifest as hypomyelination and cerebral vacuole development in several rodent species. In addition to a membrane isoform, the human Atm locus generates by alternative splicing a secreted form corresponding to the entire ectodomain that then circulates at high concentration in the periphery, released in part by activated T lymphocytes. We report here that the secreted form mRNA is downregulated throughout representative discrete regions of the human brain while membrane attractin mRNA is well represented, resulting in the apparent absence of secreted attractin protein in cerebrospinal fluid (CSF). Transcription of attractin secreted form mRNA is strongly downregulated upon differentiation of a human cortical neuron-derived cell line (HCN-1A) to a mature neuron phenotype in response to nerve growth factor. Recombinant secreted attractin disrupts neurite formation by differentiated HCN-1A cells, resulting in higher levels of branching with shorter processes. This effect is duplicated by anti-attractin and by human serum but not by human serum depleted of attractin or by CSF We propose that inappropriate expression of secreted attractin in the CNS blocks membrane attractin function and that its presence, either by leakage from the periphery, aberrant transcription, or release from inflammatory foci may affect neuron extracellular interactions leading to neurodegeneration in the human.

Constitutive activation of the LH receptor is associated with an alteration in the conformation of the ectodomain

The human LH receptor (hLHR) consists of a heptahelical segment prototypical of G protein-coupled receptors and a large ectodomain. The binding of hormone to the ectodomain or the presence of activating mutations stabilize the hLHR in an active conformation. Although it has been inferred that activation of the hLHR involves conformational alterations, direct evidence supporting this has not been reported. We have addressed this issue by comparing the protease sensitivity of the wild-type hLHR as compared to three activating mutants of the hLHR. Our data demonstrate that the ectodomains of the activating hLHR mutants are more susceptible to proteolysis as compared to the wild-type hLHR. As such, they provide the first experimental data demonstrating that the conformations of the active and inactive states of the hLHR are distinct and suggest that activation of the hLHR (at least as induced by mutations) involves a tighter interaction of the ectodomain with the heptahelical segment of the receptor as opposed to a release of the ectodomain from the heptahelical segment.

The Search for α-Secretase and its Potential as a Therapeutic Approach to Alzheimer's Disease

In the nonamyloidogenic processing pathway the Alzheimer s amyloid precursor protein (APP) is proteolytically cleaved by alpha-secretase. As this cleavage occurs at the Lys16-Leu17 bond within the amyloid beta domain, it prevents deposition of intact amyloidogenic peptide. In addition, the large ectodomain (sAPP(alpha)) released by the action of alpha-secretase has several neuroprotective properties. Studies with a range of hydroxamic acid-based compounds, such as batimastat, indicate that alpha-secretase is a zinc metalloproteinase, and members of the adamalysin family of proteins, TACE, ADAM10 and ADAM9, all fulfil some of the criteria required of alpha-secretase. APP is constitutively cleaved by alpha-secretase in most cell lines. However, on stimulation with muscarinic agonists or activators of protein kinase C, such as phorbol esters, the alpha-secretase cleavage of APP is up-regulated. The constitutive alpha-secretase activity is primarily at the cell surface, while the regulated activity is predominantly located within the Golgi. The beneficial action of cholinesterase inhibitors may in part be due to activation of muscarinic receptors, resulting in an up-regulation of alpha-secretase. Other agents can also increase the nonamyloidogenic cleavage of APP including estrogen, testosterone, various neurotransmitters and growth factors. As the alpha-secretase cleavage of APP both precludes the deposition of the amyloid beta peptide and releases the neuroprotective sAPP(alpha), pharmacological up-regulation of alpha-secretase may provide alternative therapeutic approaches for Alzheimer s disease.

Very Large G Protein-coupled Receptor-1, the Largest Known Cell Surface Protein, Is Highly Expressed in the Developing Central Nervous System

We previously identified a member of the G protein-coupled receptor family, very large G protein-coupled receptor-1 (VLGR1). VLGR1 has a large ectodomain containing multiple calcium exchanger beta repeats that resemble regulatory domains of sodium-calcium exchanger proteins. Similar repeats are found in the extracellular aggregation factor of marine sponges, which mediates species-specific cell aggregation. We now report that the protein encoded by the originally described human cDNA (now termed VLGR1a) is, in fact, at 1967 amino acids, the smallest of three expressed human isoforms. It is encoded by an alternative transcript that begins within intron 64 of the VLGR1 gene. The longest gene product, VLGR1b, is 6307 amino acids (6298 amino acids in mice) due to a much larger ectodomain containing 35 calcium exchanger beta repeats and a pentraxin homology domain. VLGR1b is apparently the largest known cell surface protein. The VLGR1 gene comprises 90 exons and is >600 kb long. In situ hybridization studies with mouse embryo sections show that high level expression of VLGR1 is restricted to the developing central nervous system and eye. Strong expression in the ventricular zone, home of neural progenitor cells during embryonal neurogenesis, suggests a fundamental role for VLGR1 in the development of the central nervous system.

Lack of Cytosolic and Transmembrane Domains of Type XIII Collagen Results in Progressive Myopathy

Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.

Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.

High-level expression of a functional single-chain human chorionic gonadotropin-luteinizing hormone receptor ectodomain complex in insect cells.

Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR). Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors. Overexpression of the ECD of the receptor has been attempted in various expression systems. Prokaryotic expression does not yield properly folded ECD. Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded. Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain). Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor. Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 microg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems. The protein was purified and binds exogenous (125)I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone. Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.

High-Level Expression of a Functional Single-Chain Human Chorionic Gonadotropin-Luteinizing Hormone Receptor Ectodomain Complex in Insect Cells

Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR). Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors. Overexpression of the ECD of the receptor has been attempted in various expression systems. Prokaryotic expression does not yield properly folded ECD. Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded. Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain). Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor. Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 μg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems. The protein was purified and binds exogenous 125I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone. Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan.

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-A resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily beta-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells.

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

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.