Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.     

Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling [22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman [7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.     

Calcium-dependent induction of novel proteins by abscisic acid in wheat aleurone tissue of different developmental stages

Aleurone tissue of mature wheat (Triticum aestivum L. cv. Sappo) grains make novel polypeptides in response to abscisic acid (ABA), but only in the presence of Ca²⁺. Effects of ABA plus Ca²⁺ include up- and down-modulation of other polypeptides. The ABA-induced polypeptides appear not to be the 21-kilodalton (kDa) amylase inhibitor which has been reported to be ABA-inducible in barley.Aleurone tissue from developing grains of different ages failed to respond to ABA plus Ca²⁺ in any way. Endogenous ABA levels were determined by monoclonal radioimmunoassay in developing, mature, and sensitised developing tissues. The ABA level rose to a maximum at 35 days post anthesis but was not detectable in mature cells. Developing layers sensitised to gibberellic acid (GA) showed decreased levels of ABA, similar to those in mature tissue, concurrent with acquired responsiveness to GA in respect of its induction of -amylase. However, these sensitised cells still remained non-responsive to added ABA in terms of modulation of polypeptide pattern, though they did respond to ABA in the blocking of GA-induced -amylase production. The role of protein phosphorylation in signal transduction was examined. The implications of these findings are discussed with reference to the role of ABA in developing and mature aleurone cells.Abbreviations ABA Abscisic acid - dpa days post anthesis - GA₃ Gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid