Prem Yadav

Three dimensional atomic model and experimental validation for the ATP-Regulated Module (ARM) of the atrial natriuretic factor receptor guanylate cyclase.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Purification and mass spectrometric analysis of the κ opioid receptor

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat kappa-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The Kd for [3H]diprenorphine was 1.1+/-0.2 nM, and the Bmax was 2.6+/-0.4 pmol/mg. Dynorphin A (1-13), U69,593 and naloxone competitively inhibited [3H]diprenorphine binding with Ki values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [35S]GTPgammaS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-beta-D-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.

ATP allosteric activation of atrial natriuretic factor receptor guanylate cyclase: ATP regulation of ANF-RGC

Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC) is the receptor and the signal transducer of two natriuretic peptide hormones, ANF and BNP. It is a single transmembrane-spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ARM) juxtaposed to the C-terminal side of the transmembrane domain of ANF-RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C-terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF-RGC must first be phosphorylated by, yet unidentified, protein kinase. This initial step is obligatory in ANF signaling of ANF-RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF-RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two-decade long debate on the role of ATP in ANF-RGC signaling. In addition, this study demonstrates that the mechanisms of allosteric modification of ANF-RGC by staurosporine and AMP-PNP, a non-hydrolyzable analogue of ATP are nearly (or totally) identical.Atrial natriuretic factor receptor guanylate cyclase (ANF‐RGC) is the receptor and the signal transducer of two natriuretic peptide hormones: atrial natriuretic factor and brain natriuretic peptide. It is a single transmembrane‐spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ATP‐regulated module) juxtaposed to the C‐terminal side of the transmembrane domain of ANF‐RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C‐terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF‐RGC must first be phosphorylated by an as yet unidentified protein kinase. This initial step is obligatory in atrial natriuretic factor signaling of ANF‐RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF‐RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two‐decade long debate on the role of ATP in ANF‐RGC signaling. In addition, the present study demonstrates that the mechanisms of allosteric modification of ANF‐RGC by staurosporine and adenylyl‐imidodiphosphate, a nonhydrolyzable analog of ATP, are almost (or totally) identical.

ATP Allosteric Activation of ANF-RGC

Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC) is the receptor and the signal transducer of two natriuretic peptide hormones, ANF and BNP. It is a single transmembrane-spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ARM) juxtaposed to the C-terminal side of the transmembrane domain of ANF-RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C-terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF-RGC must first be phosphorylated by, yet unidentified, protein kinase. This initial step is obligatory in ANF signaling of ANF-RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF-RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two-decade long debate on the role of ATP in ANF-RGC signaling. In addition, this study demonstrates that the mechanisms of allosteric modification of ANF-RGC by staurosporine and AMP-PNP, a non-hydrolyzable analogue of ATP are nearly (or totally) identical.Atrial natriuretic factor receptor guanylate cyclase (ANF‐RGC) is the receptor and the signal transducer of two natriuretic peptide hormones: atrial natriuretic factor and brain natriuretic peptide. It is a single transmembrane‐spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ATP‐regulated module) juxtaposed to the C‐terminal side of the transmembrane domain of ANF‐RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C‐terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF‐RGC must first be phosphorylated by an as yet unidentified protein kinase. This initial step is obligatory in atrial natriuretic factor signaling of ANF‐RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF‐RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two‐decade long debate on the role of ATP in ANF‐RGC signaling. In addition, the present study demonstrates that the mechanisms of allosteric modification of ANF‐RGC by staurosporine and adenylyl‐imidodiphosphate, a nonhydrolyzable analog of ATP, are almost (or totally) identical.