Phospho-ANP Recptor A

Alternate nomenclature: Arterial Natriuretic peptide receptor A, NPR-A, gunaylate ctyclase A, GC-A, .

Cyclic GMP (cGMP), a key messenger in several signal transduction pathways, the intracellular levels of cGMP ate maintained by the activity of opposing enzymes: synthesizing guanylate cyclases (GC) and hydrolyzing phosphodiesterases (PDEs). The synthesizing enzymes (GCs) are found in two forms: cytosolic (soluble) and membrane-bound (particulate), while they share similar structural characteristics, they differ in their mechanisms of physiological regulations. Most importantly, sGC contains a heme group and binds NO that activates the enzyme, while particulate GC is stimulated by natriuretic peptides. In response to G-protein couples receptor stimulation, the cGMP can be produced from GTP by either cytoplasmic, soluble guanylate cyclase (sGC) are heterodimers (a and b polypeptide chains), that are stimulated by nitric oxide and carbon monoxide or by particulate membrane-bound guanylyl cyclases which are activated by a complex mechanism by natriuretic peptides. Particulate GC (PGCs) has 7 different isoforms, PGC-A through PGC-G and are expressed in most tissues in isoform specific manner. There is significant structural homology among various PGCs, there is a large N-terminal extracellular domain (ECD), a single TMD and a large intracellular domain with protein kinase activity (KLD), a C-terminal catalytic domain (CD) and in between is a dimerization domain (DD). Both PGC-A and PGC-B are phosphorylated at Serine residues in the KLD (1). Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents (1). It has been shown that significant number hippocampal astrocytes (67%) contained both soluble and particulate guanylate cyclases in the same cell (2).

Arterial natriuretic peptide (ANP) is a cardiac hormone that regulates several important physiological functions including fluid balance, vascular smooth muscle tone, and cell growth (1). The ANP binds to ANP clearance receptor (form its removal form circulation) and an ANP receptor type A (ANPR-A) for signal transduction. The ANPR-A has 4 distinct structural/functional domains, N-terminal ligand binding domain,. A single TMD, kinase homology domain (KHD), and a C-terminal guanylate cycalse domain. Therefore it is also known as a member of ever expanding guanylate cycalse A (2). ANPR-A exist as a higher-ordered homomeric structure in absence of ligand, binding of ANP in the presence of ATP, ATP decrease the ANP binding to ANPR-A, causes a significant increase in guanylate cyclase activity immediately followed by desensitization of ANPR. It is believed that ANPR-A is constitutively phosphoryalated like many serpentine receptors and de-phosphorylation caused desensitization of ANPR. There are four serine and two threonine residues located near the putative ATP binding site on ANPR-A that are phosphorylated when ANPR-A is expressed in un-stimulated HEK293 cells (3). Mutations of these sites to alanine resulted in decreases phosphorylation state and reduced GTPase activity (3).

Phospho-specific NPRA antibodies were generated against synthetic peptide corresponding to the S497, T500 and S510. The peptide containing phospho serine and threonine is post-synthetically modified before conjugation to a carrier protein. The phospho-specific antibodies to ANPR-A are selected over differential affinity chromatography on immobilized phosphor and non-phospho ligand matrices. The affinity purified antibodies do not have any residual pan-activity or affinity for non-phosphorylated protein. FabGennix Inc. also provides antigenic peptide for blocking experiment. FabGennix provides custom antibody development services for investigators requiring application specific high affinity antibodies in different hosts. For a complete list of all FabGennix antibodies please visit www.FabGennix.com.

Immunogen:      Synthetic peptide taken form ANPR-A receptor corresponding to aa rsags(p)rlt(p)lsgrgsnygs(p)l. Peptide antigenic sequence was covalently modified to achieve desired antigenicity before conjugating to a carrier protein that is used to generate antibodies in rabbit.

Concentration:     PANPR-140AP and PANPRA-FITC antibodies have 0.75-0.76 mg/ml of antibody in stabilization buffer

Applications:        PANPRA-140AP is tested for WB and IHC application at 1:500 dilution. Other applications for this antibody have not been tested. WB: > 1:500; IMM & i.p pull-down assays: n.d; IHC n.d. (Antibody dilutions for this antibody is for reference only, investigators are expected to determine the optimal conditions). Investigators using this antibody in applications not listed here can request for a complimentary sample.                                            

Protocols:           Standard protocol for various applications (WB, IMM, IHC) of this antibody is provided with the product specification sheet, however, FabGennix Inc. strongly recommends investigators to optimize conditions for use of this antibody in their laboratories.