Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

Majumdar, Ritankar; Railkar, Reema; Dighe, Rajan R.

doi:10.1016/j.febslet.2012.01.052

Cited by 11 publications

(7 citation statements)

References 19 publications

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“…This hypothesis is consistent with the results reported for an activating antibody 13B1 of the hinge region of LHCGR ( 42 ), which interacts via a discontinuous sequence epitope comprising the N-terminal end of the exon10-region (Cb-2: 291–298), at Cb-3 with the signaling-sensitive tyrosine region ( 30 – 33 ), and two further residues ( 37 , 39 ). It seems feasible that these three discontinuous sequence regions will be assembled close together as a fully accessible patch on the surface of the hinge region.…”

Section: Discussionsupporting

confidence: 92%

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor’s Extracellular Hinge Region

et al. 2015

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The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) – secreted by the placenta, and lutropin (LH) – produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor’s leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

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Section: Discussionsupporting

confidence: 92%

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor’s Extracellular Hinge Region

et al. 2015

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“…Although specific Abs against variety of antigens, including some GPCRs, have been developed using phage display technology [ 50 , 51 , 52 ], the most common method of generating Abs, by immunizing animals with target protein, has been generally unsuccessful in the case of GPCRs. In fact, most of the available anti-GPCR Abs are polyclonal Abs purified from the serum of animals immunized with synthetic peptides corresponding to amino acid sequences within the amino (extracellular)-terminal and carboxyl (intracellular)-terminal domains and the extra- and intra-cellular loops of the GPCRs.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1

et al. 2017

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Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

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“…When tested in vitro , antiA and antiB immunoglobulins behaved as antagonists for FSH binding and for cAMP production, whereas antiC immunoglobulins did not compete for hormone binding but displayed agonist activity on FSHR-mediated cAMP response (169). Studies using polyclonal and monoclonal antibodies or scFv fragments specific of the hinge region of FSHR, LHR, or TSHR, while not affecting hormone binding, all revealed agonistic activities, unequivocally establishing the role of the hinge region in the activation of these receptors (170–172). More recently, recombinant nanobodies capable of specifically recognizing FSHR and of inhibiting cAMP accumulation have been identified (173).…”

Section: Biased Signaling At the Fshrmentioning

confidence: 95%

Biased Signaling and Allosteric Modulation at the FSHR

et al. 2019

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Knowledge on G protein-coupled receptor (GPCRs) structure and mechanism of activation has profoundly evolved over the past years. The way drugs targeting this family of receptors are discovered and used has also changed. Ligands appear to bind a growing number of GPCRs in a competitive or allosteric manner to elicit balanced signaling or biased signaling (i.e., differential efficacy in activating or inhibiting selective signaling pathway(s) compared to the reference ligand). These novel concepts and developments transform our understanding of the follicle-stimulating hormone (FSH) receptor (FSHR) biology and the way it could be pharmacologically modulated in the future. The FSHR is expressed in somatic cells of the gonads and plays a major role in reproduction. When compared to classical GPCRs, the FSHR exhibits intrinsic peculiarities, such as a very large NH2-terminal extracellular domain that binds a naturally heterogeneous, large heterodimeric glycoprotein, namely FSH. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα subunits. G protein-coupled receptor kinases and β-arrestins are also recruited to this receptor and account for its desensitization, trafficking, and intracellular signaling. Different classes of pharmacological tools capable of biasing FSHR signaling have been reported and open promising prospects both in basic research and for therapeutic applications. Here we provide an updated review of the most salient peculiarities of FSHR signaling and its selective modulation.

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Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

Cited by 11 publications

References 19 publications

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor’s Extracellular Hinge Region

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor’s Extracellular Hinge Region

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1

Biased Signaling and Allosteric Modulation at the FSHR

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