Carboxyl-terminal Tail Research Articles

Contribution of the Carboxyl Terminus of the VPAC1 Receptor to Agonist-induced Receptor Phosphorylation, Internalization, and Recycling

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese Hamster Ovary (CHO)-K1 cells

A type I transmembrane protein, Mat-8 (FXYD-3), was tagged with fluorescent protein, Discosoma red fluorescent protein, at the carboxyl terminal cytoplasmic tail, and stably expressed in Chinese Hamster Ovary (CHO)-K1 cells. The fluorescence signal was distributed in intracellular membranes, being not only detected around the nuclear envelope but also partly overlapping with an endoplasmic reticulum marker. Subcellular fractionation by density gradient centrifugation supported this partial overlapping. The spherical structures observed were not colocalized with markers for lysosomes, endosomes, and Golgi bodies, suggesting that Mat-8 is distributed in a distinct endoplasmic reticulum region and the nuclear envelope after synthesis on membrane-bound ribosomes.

The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation

In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.

Selective Role of a Distinct Tyrosine Residue on Tie2 in Heart Development and Early Hematopoiesis

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development.

Saccharomyces cerevisiae Histone H2A Ser122 Facilitates DNA Repair

DNA repair takes place in the context of chromatin. Recently, it has become apparent that proteins that make up and modulate chromatin structure are involved in the detection and repair of DNA lesions. We previously demonstrated that Ser129 in the carboxyl-terminal tail of yeast histone H2A is important for double-strand-break responses. By undertaking a systematic site-directed mutagenesis approach, we identified another histone H2A serine residue (Ser122) that is important for survival in the presence of DNA-damaging agents. We show that mutation of this residue does not affect DNA damage-dependent Rad53 phosphorylation or G(2)/M checkpoint responses. Interestingly, we find that yeast lacking H2A S122 are defective in their ability to sporulate. Finally, we demonstrate that H2A S122 provides a function distinct from that of H2A S129. These data demonstrate a role for H2A S122 in facilitating survival in the presence of DNA damage and suggest a potential role in mediating homologous recombination. The distinct roles of H2A S122 and S129 in mediating these responses suggest that chromatin components can provide specialized functions for distinct DNA repair and survival mechanisms and point toward the possibility of a complex DNA damage responsive histone code.

STAT5A interacts with and is phosphorylated upon activation of the µ‐opioid receptor

Signal Transducers and Activators of Transcription (STATs) are transcription factors shown to be activated by G protein-coupled receptors. In the present study, we demonstrate that acute morphine or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) exposure of COS-7 cells transiently transfected with the micro-opioid receptor and STAT5A, leads to receptor-dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the micro-opioid receptor with micro-opioid agonists results in the transcriptional activation of a STAT-responsive reporter gene. Pertussis toxin has no effect on the level of STAT5A phosphorylation, while the Src inhibitor PP1 abolishes opioid-dependent STAT5A phosphorylation. All three opioid receptor subtypes -micro, delta and kappa- share the conserved motif YXXL (amino-acids 336-339 for the micro-opioid receptor), known to be critical for STAT5A/5B binding. Co-immunoprecipitation and pull-down experiments using a GST-carboxyl-terminal tail of the micro-opioid receptor and rat brain, or COS-7 cell cytosolic extracts, demonstrate the direct binding of STAT5A to this region. Mutation of the Y336 to alanine does not prevent STAT5A binding, whereas deletion of the entire putative STAT5A binding site YXXL abolishes STAT5A interaction to the carboxyl-terminal tail of the micro-opioid receptor. Collectively, our results demonstrate the association of STAT5A with the micro-opioid receptor and reveal novel signalling pathways in the regulation of transcription by the micro-opioid receptor.

Lactisole Interacts with the Transmembrane Domains of Human T1R3 to Inhibit Sweet Taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.

Molecular Identification and Characterization of Xenopus Egg Uroplakin III, an Egg Raft-associated Transmembrane Protein That Is Tyrosine-phosphorylated upon Fertilization

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

AP-1 and AP-3 Facilitate Lysosomal Targeting of Batten Disease Protein CLN3 via Its Dileucine Motif

CLN3 is a transmembrane protein with a predominant localization in lysosomes in non-neuronal cells but is also found in endosomes and the synaptic region in neuronal cells. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinosis or Batten disease, which currently is the most common cause of childhood dementia. We have recently reported that the lysosomal targeting of CLN3 is facilitated by two targeting motifs: a dileucine-type motif in a cytoplasmic loop domain and an unusual motif in the carboxyl-terminal cytoplasmic tail comprising a methionine and a glycine separated by nine amino acids (Kyttala, A., Ihrke, G., Vesa, J., Schell, M. J., and Luzio, J. P. (2004) Mol. Biol. Cell 15, 1313-1323). In the present study, we investigated the pathways and mechanisms of CLN3 sorting using biochemical binding assays and immunofluorescence methods. The dileucine motif of CLN3 bound both AP-1 and AP-3 in vitro, and expression of mutated CLN3 in AP-1- or AP-3-deficient mouse fibroblasts showed that both adaptor complexes are required for sequential sorting of CLN3 via this motif. Our data indicate the involvement of complex sorting machinery in the trafficking of CLN3 and emphasize the diversity of parallel and sequential sorting pathways in the trafficking of membrane proteins.

Subtype-Specific Sorting of the ETAEndothelin Receptor by a Novel Endocytic Recycling Signal for G Protein-Coupled Receptors

We have previously reported that endocytic sorting of ET(A) endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxyl-terminal recycling motif of the ET(A) receptor. Assay of truncation mutants of the ET(A) receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel beta-strand structures (beta-finger). One of these segments was the beta-finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domain-binding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ET(A) receptor was predicted to attain a beta-finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative beta-finger conformation or PDZ ligand function prevented recycling of the ET(A) receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET(A) receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Myosin V attachment to cargo requires the tight association of two functional subdomains

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle–specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.

Dermal fibroblasts in Hutchinson-Gilford progeria syndrome with the lamin A G608G mutation have dysmorphic nuclei and are hypersensitive to heat stress

BackgroundHutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT), within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein.ResultsWe studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress.ConclusionDermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic abnormalities in the disease.

Developmental expression of the High Mobility Group B gene in the amphioxus, Branchiostoma belcheri tsingtauense

High-Mobility Group (HMG) B proteins are abundant and highly conserved non-histone proteins, which play an important architectural role in the assembly of nucleoprotein complexes and in the Regulation of transcription. These proteins have also been shown to play key roles during embryonic development and cell differentiation. Here we report a full-length cDNA sequence of the HMG protein, AmphiHMGB from Amphioxus. Sequence analysis indicates that this putative AmphiHMGB protein contains four domains: HMG-box A, HMG-box B, basic region, acidic carboxyl-terminal tail and a linker. Phylogenetic analysis suggests that AmphiHMGB falls outside the vertebrate clade. HMGB gene duplication occurred near the base of the vertebrate gene Clade. The dynamic expression of AmphiHMGB during embryonic development reveals for the first time that it may involve differentiation of neural ectoderm, mesoderm and endoderm in this animal.

Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

Epstein-Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lines (AGS and SCC68) or the human embryonic kidney cell line 293-P. Fusion with human embryonic kidney 293-P cells was greater than fusion observed with B cells, indicative of an important role for cell contact. An antibody directed against the gH and gL complex inhibited epithelial cell fusion. Increased surface expression of gB alone as a result of truncations or point mutants in the carboxyl-terminal tail allowed gB-mediated fusion with epithelial cells, albeit at a lower level than with coexpression of gB, gH, and gL. Overall, gB appears to be the critical component for EBV glycoprotein-mediated cell fusion.

Molecular and Biochemical Characterization of the Skp2-Cks1 Binding Interface

SCF(Skp2) is a multisubunit E3 ubiquitin ligase responsible for ubiquitination of cell cycle inhibitor p27. Ubiquitination of p27 requires an adapter protein, Cks1, to be in direct association with Skp2. The exact interface between Skp2 and Cks1 has not been elucidated. Here we have reported the definition of the critical functional interface between Skp2 and Cks1. We have identified eight amino acid residues in two discrete regions of Skp2 that are engaged in Cks1 binding. Mutation of any of these eight residues alone or in combination results in the loss of Cks1 association and negates Skp2-dependent p27 ubiquitination. These eight amino acid residues map on the same side of the Skp2 structure and likely constitute a functional binding surface for Cks1. Four of the eight amino acid residues are located in the largely unstructured carboxyl-terminal tail region of Skp2. These results uncovered the specificity of the Skp2-Cks1 interaction and reveal a critical function for the structurally flexible carboxyl-terminal tail region of Skp2 in Cks1 recognition and substrate ubiquitination.

Identification of Amino Acid Residues That Direct Differential Ligand Selectivity of Mammalian and Nonmammalian V1a Type Receptors for Arginine Vasopressin and Vasotocin: INSIGHTS INTO MOLECULAR COEVOLUTION OF V1a TYPE RECEPTORS AND THEIR LIGANDS

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.

Membrane-Proximal Region of the Carboxyl Terminus of the Gonadotropin-Releasing Hormone Receptor (GnRHR) Confers Differential Signal Transduction between Mammalian and Nonmammalian GnRHRs

Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.

Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans

In human beings, two forms of GnRH, termed GnRH-I and GnRH-II, encoded by separate genes have been identified. Although these hormones share comparable cDNA and genomic structures, their tissue distribution and regulation of gene expression are significantly dissimilar. The actions of GnRH are mediated by the GnRH receptor, which belongs to a member of the rhodopsin-like G protein-coupled receptor superfamily. However, to date, only one conventional GnRH receptor subtype (type I GnRH receptor) uniquely lacking a carboxyl-terminal tail has been found in the human body. Studies on the transcriptional regulation of the human GnRH receptor gene have indicated that tissue-specific gene expression is mediated by differential promoter usage in various cell types. Functionally, there is growing evidence showing that both GnRH-I and GnRH-II are potentially important autocrine and/or paracrine regulators in some extrapituitary compartments. Recent cloning of a second GnRH receptor subtype (type II GnRH receptor) in nonhuman primates revealed that it is structurally and functionally distinct from the mammalian type I receptor. However, the human type II receptor gene homolog carries a frameshift and a premature stop codon, suggesting that a full-length type II receptor does not exist in humans.