scholarly journals FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function

2015 ◽  
Vol 54 (3) ◽  
pp. 193-204 ◽  
Author(s):  
Antara A Banerjee ◽  
Madhavi Dupakuntla ◽  
Bhakti R Pathak ◽  
Smita D Mahale
Keyword(s):  
Amino Acids ◽  
Hek293 Cells ◽  
Fsh Receptor ◽  
Receptor Function ◽  
Extracellular Loop ◽  
Camp Production ◽  
Transfected Cells ◽  
Erk Phosphorylation ◽  
Loop 2

The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH–FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L501F mainly and I505V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L501F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.

scholarly journals Probing the role of loop 2 in Ras function with unnatural amino acids.

1993 ◽  
Vol 90 (21) ◽  
pp. 10145-10149 ◽  
Author(s):  
H. H. Chung ◽  
D. R. Benson ◽  
V. W. Cornish ◽  
P. G. Schultz
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Loop 2

scholarly journals Role of the Second Extracellular Loop of Human C3a Receptor in Agonist Binding and Receptor Function

1999 ◽  
Vol 274 (14) ◽  
pp. 9721-9728 ◽  
Author(s):  
Ta-Hsiang Chao ◽  
Julia A. Ember ◽  
Meiying Wang ◽  
Yolanda Bayon ◽  
Tony E. Hugli ◽  
...  
Keyword(s):  
Receptor Function ◽  
Agonist Binding ◽  
Extracellular Loop

Abstract 273: Phosphoinositide 3-Kinase γ Regulates Cardio-protective ERK by Kinase Independent Mechanism

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
George Jolly ◽  
Rohit Anand ◽  
Sathyamangla V Naga Prasad
Keyword(s):  
Tyrosine Kinases ◽  
Hek293 Cells ◽  
Independent Function ◽  
Signaling Mechanism ◽  
Erk Activation ◽  
Cellular Processes ◽  
Erk Phosphorylation ◽  
Phosphoinositide 3 Kinase ◽  
Sirna Knockdown

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.

scholarly journals In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity

2020 ◽  
Vol 4 (5) ◽  
Author(s):  
Teresa Zariñán ◽  
Viktor Y Butnev ◽  
Rubén Gutiérrez-Sagal ◽  
José Luis Maravillas-Montero ◽  
Iván Martínez-Luis ◽  
...  
Keyword(s):  
Hek293 Cells ◽  
Signaling Cascades ◽  
Fsh Receptor ◽  
Functional Selectivity ◽  
Camp Production ◽  
Human Pituitary ◽  
Lower Extent ◽  
Gs Protein ◽  
Ca2 Channels

Abstract FSH exists as different glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH24) and hypo-glycosylated FSH (FSH18/21) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH18/21 and FSH24) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH18/21, FSH24, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH18/21 followed by eFSH, recFSH, and FSH24. In β-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH18/21 exhibited a preference toward β-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of β-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH18/21. Concurrent silencing of β-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and β-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca2+ (iCa2+) accumulation through both influx from Ca2+ channels and release from intracellular stores; however, iCa2+ in response to FSH18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa2+ accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.

scholarly journals Relationship between Thyrotropin Receptor Hinge Region Proteolytic Posttranslational Modification and Receptor Physiological Function

2011 ◽  
Vol 25 (1) ◽  
pp. 184-194 ◽  
Author(s):  
Sepehr Hamidi ◽  
Chun-Rong Chen ◽  
Yumiko Mizutori-Sasai ◽  
Sandra M. McLachlan ◽  
Basil Rapoport
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Hormone Receptor ◽  
Hinge Region ◽  
Receptor Function ◽  
Glycoprotein Hormone ◽  
Glycoprotein Hormone Receptor ◽  
Functional Alteration

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261–412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening ‘C-peptide’ region. Experimentally, hinge region amino acids 317–366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function (‘minimal hinge’) are lacking. Using as a template the functionally normal TSHR with residues 317–366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307–371 deletion) was also tolerated without functional alteration, as was deletion of residues 303–366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377–384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303–366 (64 residues) or 307–371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.

scholarly journals Characterization of feline ASCT1 and ASCT2 as RD-114 virus receptor

2013 ◽  
Vol 94 (7) ◽  
pp. 1608-1612 ◽  
Author(s):  
Sayumi Shimode ◽  
Rie Nakaoka ◽  
Hiroko Shogen ◽  
Takayuki Miyazawa
Keyword(s):  
Endogenous Retrovirus ◽  
Amino Acid Transporters ◽  
Extracellular Loop ◽  
Virus Receptor ◽  
Metabolic Role ◽  
Virus Receptors ◽  
Sodium Dependent ◽  
Loop 2

RD-114 virus is a replication-competent feline endogenous retrovirus (ERV). RD-114 virus had been thought to be xenotropic; however, recent findings indicate that RD-114 virus is polytropic and can infect and grow efficiently in feline cells. Receptor(s) for RD-114 virus has not been identified and characterized in cats. In this study, we confirmed that two feline sodium-dependent neutral amino acid transporters (ASCTs), fASCT1 and fASCT2, function as RD-114 virus receptors. By chimeric analyses of feline and murine ASCTs, we revealed that extracellular loop 2 of both fASCT1 and fASCT2 determines the susceptibility to RD-114 virus. Further, we revealed ubiquitous expression of these genes, consistent with the general metabolic role of the ASCT molecules. Our study indicates that RD-114 virus may reinfect tissues and cells in cats, once the virus is activated. Implications of the involvement of RD-114 virus in feline oncogenesis are also discussed.

scholarly journals Amphotropic Murine Leukemia Virus Entry Is Determined by Specific Combinations of Residues from Receptor Loops 2 and 4

1999 ◽  
Vol 73 (4) ◽  
pp. 3169-3175 ◽  
Author(s):  
Mikkel D. Lundorf ◽  
Finn S. Pedersen ◽  
Bryan O’Hara ◽  
Lene Pedersen
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Membrane Topology ◽  
Chimeric Proteins ◽  
Receptor Function ◽  
Amphotropic Murine Leukemia Virus ◽  
Extracellular Loops ◽  
Loop 2 ◽  
Murine Leukemia

ABSTRACT Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.

scholarly journals Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1038-1047 ◽  
Author(s):  
Isabelle Mouro-Chanteloup ◽  
Anne Marie D'Ambrosio ◽  
Pierre Gane ◽  
Caroline Le Van Kim ◽  
Virginie Raynal ◽  
...  
Keyword(s):  
Cell Surface ◽  
K562 Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Cmv Promoter ◽  
Transfected Cells

Abstract In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation

2012 ◽  
Vol 362 (1-2) ◽  
pp. 60-68 ◽  
Author(s):  
Madhavi Dupakuntla ◽  
Bhakti Pathak ◽  
Binita Sur Roy ◽  
Smita D. Mahale
Keyword(s):  
Receptor Activation ◽  
Fsh Receptor ◽  
Extracellular Loop ◽  
Loop 2
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