scholarly journals Conserved Amino Acid Residues that Are Important for Ligand Binding in the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor Are Required for High Potency of GnRH II at the Type II GnRH Receptor

2007 ◽  
Vol 21 (1) ◽  
pp. 281-292 ◽  
Author(s):  
Sipho Mamputha ◽  
Zhi-liang Lu ◽  
Roger W. Roeske ◽  
Robert P. Millar ◽  
Arieh A. Katz ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Gnrh Receptor ◽  
Type I ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Binding Assays ◽  
Antagonist Activity ◽  
Conserved Residues ◽  
Gnrh Receptors ◽  
Mutant Receptors

Abstract GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the C⋕O group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.

scholarly journals Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus

2006 ◽  
Vol 36 (3) ◽  
pp. 601-610 ◽  
Author(s):  
M R Silver ◽  
S A Sower
Keyword(s):  
Intact Cell ◽  
Functional Characterization ◽  
Kinetic Studies ◽  
Receptor Activation ◽  
Gnrh Receptor ◽  
Type I ◽  
Type Ii ◽  
Binding Assays ◽  
Gnrh Receptors ◽  
Series Of Experiments

The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependant manner when treated with either lamprey GnRH-I (LogEC50 −6.57±0.15) or lamprey GnRH-III (LogEC50 −8.29±0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708±0.245 nM)=chicken GnRH-II (Ki 0.765±0.160 nM) > mammalian GnRH (Ki 12.9±1.96 nM) > dAla6Pro9NEt mammalian GnRH (Ki 21.6±9.68 nM) > lamprey GnRH-I (Ki 118.0±23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependant internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.

scholarly journals Type II gonadotrophin-releasing hormone (GnRH-II) in reproductive biology

Reproduction ◽  
2003 ◽  
pp. 271-278 ◽  
Author(s):  
AJ Pawson ◽  
K Morgan ◽  
SR Maudsley ◽  
RP Millar
Keyword(s):  
Receptor Gene ◽  
Gnrh Receptor ◽  
Specific Gene ◽  
Type I ◽  
Type Ii ◽  
Releasing Hormone ◽  
Human Type ◽  
Gnrh Receptors ◽  
Gonadotrophin Releasing Hormone ◽  
Ligand Precursors

Humans may be particularly unusual with respect to the gonadotrophin-releasing hormone (GnRH) control of their reproductive axis in that they possess two distinct GnRH precursor genes, on chromosomes 8p11-p21 and 20p13, but only one conventional GnRH receptor subtype (type I GnRH receptor) encoded within the genome, on chromosome 4. A disrupted human type II GnRH receptor gene homologue is present on chromosome 1q12. The genes encoding GnRH ligand precursors and GnRH receptors have now been characterized in a broad range of vertebrate species, including fish, amphibians and mammals. Ligand precursors and receptors can be categorized into three phylogenetic families. Members of each family exist in primitive vertebrates, whereas mammals exhibit selective loss of ligand precursor and receptor genes. One interpretation of these findings is that each ligand-cognate receptor family may have evolved to fulfil a separate function in reproductive physiology and that species-specific gene inactivation, modification or loss may have occurred during evolution when particular roles have become obsolete or subject to regulation by a different biochemical pathway. Evidence in support of this concept is available following the characterization of the chromosomal loci encoding the human type II GnRH receptor homologue, a rat type II GnRH receptor gene remnant (on rat chromosome 18) and a mouse type II GnRH ligand precursor gene remnant (on mouse chromosome 2). Whether type I GnRH and type II GnRH peptides elicit different signalling responses in humans by activation of the type I GnRH receptor in a cell type-specific fashion remains to be shown. Recent structure-function studies of GnRH ligands and GnRH receptors and their expression patterns in different tissues add further intrigue to this hypothesis by indicating novel roles for GnRH such as neuromodulation of reproductive function and direct regulation of peripheral reproductive tissues. Surprises concerning the complexities of GnRH ligand and receptor function in reproductive endocrinology should continue to emerge in the future.

scholarly journals Tunicate Gonadotropin-Releasing Hormone (GnRH) Peptides Selectively Activate Ciona intestinalis GnRH Receptors and the Green Monkey Type II GnRH Receptor

Endocrinology ◽  
2005 ◽  
Vol 146 (9) ◽  
pp. 4061-4073 ◽  
Author(s):  
Javier A. Tello ◽  
Jean E. Rivier ◽  
Nancy M. Sherwood
Keyword(s):  
Messenger Rna ◽  
Inositol Phosphate ◽  
Ciona Intestinalis ◽  
Positive Control ◽  
Gnrh Receptor ◽  
Biologically Active ◽  
Intracellular Camp ◽  
Type Ii ◽  
Gnrh Receptors ◽  
Green Monkey

Abstract In vertebrates, GnRH binds to its receptor and stimulates predominantly Gq/11-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.

scholarly journals Amphioxus: Beginning of Vertebrate and End of Invertebrate Type GnRH Receptor Lineage

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2847-2856 ◽  
Author(s):  
Javier A. Tello ◽  
Nancy M. Sherwood
Keyword(s):  
Inositol Phosphate ◽  
Gnrh Receptor ◽  
The Other ◽  
Type I ◽  
Adipokinetic Hormone ◽  
Branchiostoma Floridae ◽  
Vertebrate Lineage ◽  
Functional Conservation ◽  
Gnrh Receptors ◽  
Amphioxus Genome

In vertebrates, activation of the GnRH receptor is necessary to initiate the reproductive cascade. However, little is known about the characteristics of GnRH receptors before the vertebrates evolved. Recently genome sequencing was completed for amphioxus, Branchiostoma floridae. To understand the GnRH receptors (GnRHR) from this most basal chordate, which is also classified as an invertebrate, we cloned and characterized four GnRHR cDNAs encoded in the amphioxus genome. We found that incubation of GnRH1 (mammalian GnRH) and GnRH2 (chicken GnRH II) with COS7 cells heterologously expressing the amphioxus GnRHRs caused potent intracellular inositol phosphate turnover in two of the receptors. One of the two receptors displayed a clear preference for GnRH1 over GnRH2, a characteristic not previously seen outside the type I mammalian GnRHRs. Phylogenetic analysis grouped the four receptors into two paralogous pairs, with one pair grouping basally with the vertebrate GnRH receptors and the other grouping with the octopus GnRHR-like sequence and the related receptor for insect adipokinetic hormone. Pharmacological studies showed that octopus GnRH-like peptide and adipokinetic hormone induced potent inositol phosphate turnover in one of these other two amphioxus receptors. These data demonstrate the functional conservation of two distinct types of GnRH receptors at the base of chordates. We propose that one receptor type led to vertebrate GnRHRs, whereas the other type, related to the mollusk GnRHR-like receptor, was lost in the vertebrate lineage. This is the first report to suggest that distinct invertebrate and vertebrate GnRHRs are present simultaneously in a basal chordate, amphioxus.

scholarly journals Classical Xanthinuria in Nine Israeli Families and Two Isolated Cases from Germany: Molecular, Biochemical and Population Genetics Aspects

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 788
Author(s):  
Hava Peretz ◽  
Ayala Lagziel ◽  
Florian Bittner ◽  
Mustafa Kabha ◽  
Meirav Shtauber-Naamati ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
Type I ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Cysteine Desulfurase ◽  
Cofactor Binding ◽  
Pathogenic Variants ◽  
Expression Studies

Classical xanthinuria is a rare autosomal recessive metabolic disorder caused by variants in the XDH (type I) or MOCOS (type II) genes. Thirteen Israeli kindred (five Jewish and eight Arab) and two isolated cases from Germany were studied between the years 1997 and 2013. Four and a branch of a fifth of these families were previously described. Here, we reported the demographic, clinical, molecular and biochemical characterizations of the remaining cases. Seven out of 20 affected individuals (35%) presented with xanthinuria-related symptoms of varied severity. Among the 10 distinct variants identified, six were novel: c.449G>T (p.(Cys150Phe)), c.1434G>A (p.(Trp478*)), c.1871C>G (p.(Ser624*)) and c.913del (p.(Leu305fs*1)) in the XDH gene and c.1046C>T (p.(Thr349Ileu)) and c.1771C>T (p.(Pro591Ser)) in the MOCOS gene. Heterologous protein expression studies revealed that the p.Cys150Phe variant within the Fe/S-I cluster-binding site impairs XDH biogenesis, the p.Thr349Ileu variant in the NifS-like domain of MOCOS affects protein stability and cysteine desulfurase activity, while the p.Pro591Ser and a previously described p.Arg776Cys variant in the C-terminal domain affect Molybdenum cofactor binding. Based on the results of haplotype analyses and historical genealogy findings, the potential dispersion of the identified variants is discussed. As far as we are aware, this is the largest cohort of xanthinuria cases described so far, substantially expanding the repertoire of pathogenic variants, characterizing structurally and functionally essential amino acid residues in the XDH and MOCOS proteins and addressing the population genetic aspects of classical xanthinuria.

scholarly journals Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1415-1422 ◽  
Author(s):  
Adam J. Pawson ◽  
Elena Faccenda ◽  
Stuart Maudsley ◽  
Zhi-Liang Lu ◽  
Zvi Naor ◽  
...  
Keyword(s):  
G Protein ◽  
Regulatory Elements ◽  
Gnrh Receptor ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Type I ◽  
Receptor Desensitization ◽  
Gnrh Receptors ◽  
Carboxyl Terminal ◽  
G Protein Coupled

Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as β-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit β-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.

Effect of mutation of a calmodulin binding site on Ca2+ regulation of inositol trisphosphate receptors

2001 ◽  
Vol 360 (2) ◽  
pp. 395-400 ◽  
Author(s):  
Xianchao ZHANG ◽  
Suresh K. JOSEPH
Keyword(s):  
Splice Variants ◽  
Binding Motif ◽  
Type I ◽  
Channel Activity ◽  
Wild Type ◽  
Binding Assays ◽  
Ip3 Receptor ◽  
Calmodulin Binding ◽  
Mutant Receptors

Several studies have shown that calmodulin (CaM) modulates d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel activity and ligand binding to IP3Rs. It has been proposed that CaM may act as the Ca2+ sensor for mediating Ca2+ inhibition of IP3R channel activity. However, the functional role of CaM binding sites and the mechanism by which CaM regulates IP3R activities remains unclear. Tryptophan at position 1577 of type I IP3R has been shown to be part of a motif that is responsible for CaM binding to IP3Rs and we have mutated this residue to alanine in the long (neuronal) and short (peripheral) SII splice variants of the type I IP3R. CaM–Sepharose binding assays using COS-7 cell lysates confirmed that the W1577A mutant in both splice variants completely eliminated CaM binding. Functional measurements of IP3-mediated 45Ca2+ fluxes indicated that there was no change in the IP3 sensitivity of the channel induced by the W1577A mutation. Such measurements also indicated that the W1577A mutants of both splice variants have a dependence on external [Ca2+] that was indistinguishable from the corresponding wild-types. Although subtle differences in the Ca2+ and CaM sensitivity of [3H]IP3 binding were noted between wild-type and mutant receptors, our data suggest that the CaM binding motif involving the W1577A locus does not play a role in Ca2+ regulation of IP3R channel activity.

Distinct localization and function of1,4,5IP3 receptor subtypes and the1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3412-3422 ◽  
Author(s):  
Samer S. El-Daher ◽  
Yatin Patel ◽  
Ashia Siddiqua ◽  
Sheila Hassock ◽  
Scott Edmunds ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Adenosine Monophosphate ◽  
Human Platelets ◽  
Surface Proteins ◽  
Receptor Subtypes ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Receptor Antibodies

Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.

Distinct localization and function of1,4,5IP3 receptor subtypes and the1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3412-3422 ◽  
Author(s):  
Samer S. El-Daher ◽  
Yatin Patel ◽  
Ashia Siddiqua ◽  
Sheila Hassock ◽  
Scott Edmunds ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Adenosine Monophosphate ◽  
Human Platelets ◽  
Surface Proteins ◽  
Receptor Subtypes ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Receptor Antibodies

Abstract Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.

A Third Lamprey Novel GnRH Receptor Similar to Gnathostome Type-II GnRH Receptors.

2010 ◽  
pp. P3-205-P3-205
Author(s):  
A Aquilina-Beck ◽  
C MacDonald ◽  
SI Kavanaugh ◽  
M Freamat ◽  
SA Sower
Keyword(s):  
Gnrh Receptor ◽  
Type Ii ◽  
Gnrh Receptors
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