Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor.

A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.     

Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [(125)I]-[N(epsilon)-(4-azidosalicyl)Lys(5)]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu(503)-Lys(517) region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu(503)-Lys(517) completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.     

Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery

In this study specific high affinity binding sites for atrial natriuretic factor (rANF(99-126] have been identified on cultured endothelial cells of bovine pulmonary artery origin (BPAEC). A time-dependent rise in cellular cGMP levels stimulated by rANF(99-126) was followed by release of the nucleotide into the incubation medium. The use of truncated, ring-deleted and linear atrial peptide analogs in competitive displacement analysis and measurement of cGMP accumulation indicated that only a minor proportion (5-11%) of the available receptor pool was of the ANF-B receptor subtype, linked to guanylate cyclase, with the remaining major proportion possibly of the ANF-C (clearance) receptor subtype. The existence of two ANF receptor subtypes in this cell culture model would suggest a significant role for the circulating peptide in modulation of pulmonary endothelial cell function, which would influence or complement its direct actions on the underlying vasculature of the pulmonary circulation.     

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro

The effects of synthetic atrial natriuretic factor (ANF) on the regulation of mouse Leydig cell steroidogenesis have been studied in vitro. ANF in nanomolar concentration increased testosterone production by more than 30-fold over basal levels. Concomitantly, cyclic guanosine monophosphate levels were increased 35-fold; cyclic adenosine monophosphate levels fell minimally (15-20%). ANF at low concentration (1 X 10(-11) M) inhibited testosterone production by luteinizing hormone-stimulated cells, while at higher concentration (greater than 2 X 10(-9) M) ANF stimulated steroidogenesis beyond the level attained by luteinizing hormone alone. These results indicate that ANF can exert stimulatory effects on testosterone steroidogenesis in vitro, and that the mechanism may involve an intracellular messenger other than cyclic adenosine monophosphate.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-125I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.     

Active cytokinins: photoaffinity labeling agents to detect binding

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N⁶-benzyladenines, -N⁶-(Δ²-isopentenyl)adenines, and -zeatins, and N⁶-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10⁻⁵ micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10⁻⁶ micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10⁻⁵ micromolar, representing a sensitivity rarely attained.     

Distinct atrial natriuretic factor receptor sites on cultured bovine aortic smooth muscle and endothelial cells

Cultured bovine aortic smooth muscle and endothelial cells each display distinct specific binding sites for radiolabeled atrial natriuretic peptide (ANF). 125I-pro-rANF (103-126)I binding to both cell types is rapid, reversible and competitive. Scatchard plots of the binding data show Bmax values of 5.5 and 0.1 - 2.1 X 10(5) sites/cell and Kd values of 2.1 and 0.3 nM for smooth muscle and endothelial cells, respectively. In addition, ANF elevates levels of cGMP substantially in both cell types at concentrations of ANF close to its Kd and Ki for binding. Sodium nitroprusside, however, has essentially no effect on cGMP levels in either cell type. These results show that distinct functionally active receptor sites for ANF exist on both vascular smooth muscle and endothelial cells.     

Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells

Previous studies from this laboratory have shown that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells without affecting cell multiplication. In an attempt to characterize the intracellular signaling systems activated by mEGF in this cell type, we examined its effects on the labeling of phosphatidylinositols in cells that had been preincubated with different radioactive precursors. Here we report that exposure of MA-10 cells to mEGF, but not other ligands that affect their differentiated function, results in an increase in the labeling of an unusual phosphatidylinositol that does not appear to be present in unstimulated cells. This phosphatidylinositol has been identified as phosphatidylinositol 3,4-bisphosphate.     

Atrial natriuretic peptide decreases contractility of cultured chick ventricular cells

To determine whether atrial natriuretic peptide (ANP) has an inotropic effect, the contractility of spontaneously beating cultured chick embryo ventricular cells was studied in response to rat-ANP (1-23) superfused at concentrations ranging from 10(-10) M to 2.5 x 10(-7) M. r-ANP reversibly decreased contractility with a threshold concentration of 10(-8) M; at the highest concentration, r-ANP decreased contractility to a moderate extent (-30 +/- 4%) r-ANP increased dose-dependently intracellular cGMP levels. Stimulation of contractility with [Ca2+], the calcium-channel agonist BAY K 8644 or isoproterenol attenuated to various degrees the inhibitory effect of r-ANP. By contrast, the inhibitory effect of r-ANP on contractility was unchanged or even enhanced after stimulation of contractility by angiotensin II. There was no difference in r-ANP-induced increase in cGMP whether cells were pre-incubated with angiotensin II or not. These results indicate that r-ANP was able to decrease contractility of cultured cardiac myocytes and suggest a preferential antagonism of the inotropic effect of angiotensin II.     

Atrial natriuretic factor: an overview

Heart atrial muscle cells in mammals are differentiated for a contractile as well as a secretory function. Through the latter, the heart plays an endocrine role; it synthesizes, stores, and releases a group of peptides collectively referred to as atrial natriuretic factor (ANF). ANF has natriuretic and hypotensive properties as well as an inhibitory effect on aldosterone and renin secretion. Thus ANF intervenes in the short- and long-term regulation of water and electrolyte balance and blood pressure. It is expected that further research in this new field will provide fresh insights into the pathophysiology of several important clinical entities and in the development of new pharmaceutical products.     

Effect of photoaffinity labeling on rabbit uterine progesterone receptor

Photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([ 3H]R5020) is an effective technique for the covalent labeling of the progesterone receptor (PR), which allows monitoring of the steroid receptor complex under denaturing conditions. The present study was initiated to evaluate whether photolabeled PR could be used also as a marker for PR under nondenaturing conditions. Accordingly, the effect of irradiation on each component of the reaction was evaluated separately. When [3H]R5020 alone was irradiated, there was a rapid (less than 5 min), light dependent destruction of [3H]R5020, as evident from increased formation of a more polar tritiated product on TLC and a concomitant decrease in the ability of the irradiated preparation to bind to PR. When rabbit uterine PR was irradiated in the absence of steroid, a gradual decrease in the binding capacity was observed, reaching 70% of the nonirradiated control in 10 min. The optimal irradiation time for covalent [3H]R5020-PR complex formation was determined by irradiation for up to 5 min, and separation of the products by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Specific labeling of proteins of Mr 116,000 and 85,000 was observed, with the rate of labeling of the two being similar, and reaching a plateau by 4 min of irradiation. The photolabeling efficiency ranged from 2 to 12%. Sucrose gradient ultracentrifugation of photolabeled PR revealed that both the irradiated sample and the nonirradiated control sedimented to the same position. Subsequent SDS-polyacrylamide gel electrophoresis of the sucrose gradient peak from the photolabeled sample showed the presence of both labeled proteins of Mr 116,000 and 85,000. In addition, photolabeled rabbit uterine PR (Mr 116,000 and 85,000) could be immunoprecipitated with a guinea pig antiserum raised against rabbit uterine PR. Analysis of the photoaffinity labeling procedure in our system revealed that the photodestruction of [3H]R5020 was very rapid. However, maximal labeling with [3H]R5020 was obtainable with minimal photodestruction of PR which suggests that photolabeled receptor can be used as a marker for PR under nondenaturing conditions.     

Atrial natriuretic factor stimulates renal dopamine uptake mediated by natriuretic peptide-type A receptor

To determine the effects of atrial natriuretic factor (ANF) on renal dopamine (DA) metabolism, 3H-DA and 3H-L-DOPA uptake by renal tubular cells was measured in experiments carried out in vitro in Sprague-Dawley rats. The receptor type involved was also analyzed. The results indicate that ANF increased at 30 min, DA uptake in a concentration-response fashion having 10 pM ANF as the threshold concentration. Conversely, the uptake of the precursor L-DOPA was not modified by the peptide. ANF effects were observed in tissues from external and juxtamedullar cortex and inner medulla. On this basis, 100 nM ANF was used to continue the studies in external cortex tissues. DA uptake was characterized as extraneuronal uptake, since 100 microM hydrocortisone blocked ANF-induced increase of DA uptake. Renal DA uptake was decreased at 0 degrees C and in sodium-free medium. The effects of ANF in these conditions were not present, confirming that renal DA uptake is mediated by temperature- and sodium-dependent transporters and that the peptide requires the presence of the ion to exhibit its actions on DA uptake. The biological natriuretic peptide type A receptor (NPR-A) mediates ANF effects, since 100 nM anantin, a specific blocker, reversed ANF-dependent increase of DA uptake. The natriuretic peptide type C receptor (NPR-C) is not involved, since the specific analogous 100 nM 4-23 ANF amide has no effect on renal DA uptake and does not alter the effects of 100 nM ANF. In conclusion, ANF stimulates DA uptake by kidney tubular cells. ANF effects are mediated by NPR-A receptors coupled to guanylate cyclase and cGMP as second messenger. The process involved was characterized as a typical extraneuronal uptake, and characterized as temperature- and sodium-dependent. This mechanism could be related to DA effects on sodium reabsorption and linked to ANF enhanced natriuresis in the kidney. The increment of endogenous DA into tubular cells, as a consequence of increased DA uptake, would permit D1 receptor recruitment and Na+,K+-ATPase activity inhibition, which results in decreased sodium reabsorption and increased natriuresis.     

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM’s structural motif, ⁶⁶⁹WTAPELL⁶⁷⁵. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the ⁶⁶⁹WTAPELL⁶⁷⁵ motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.