Identification of Cysteine-Rich Epidermal Growth Factor–Like Domain 1α (CRELD1α) as a Novel α1A-Adrenoceptor–Down-Regulating Protein and Establishment of an α1L-Adrenoceptor–Expressing Cell Line

Abstract

Abstract. Two distinct α 1 -adrenoceptor phenotypes ( α 1A -and α 1L -ARs) are known to originate from a single ADRA1A( α 1a ) gene by an as-yet-unknown mechanism. We hypothesized that an α 1a -AR-interacting protein could generate the α 1L -AR phenotype and we sought to identify such a protein and to examine its effects on the expression of α 1A and α 1L phenotypes. Cysteine-rich epidermal growth factor-like domain 1 α (CRELD1 α ) was first identified using a yeast two-hybrid approach as an α 1a -AR-interacting protein. Transfection of α 1a -AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing α 1A -ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites ( α 1L -AR). Knockdown of endogenous CHO-CRELD1 α [ α 1a -CKD( α 1A -enhanced) cells] enhanced the expression of α 1A -AR, whereas over-expression of CRELD1 α reduced α 1A -AR expression, yielding α 1a -COE( α 1L -dominant) cells expressing a high proportion (50%) of the α 1L -AR phenotype. The ligand binding and functional agonist and antagonist profiles in α 1a -CKD( α 1A -enhanced) and α 1a -COE( α 1L -dominant) cell lines were entirely in accord with the α 1A -AR and α 1L -AR phenotypes observed in intact tissues. CRELD1 α down-regulates expression of the α 1A -AR, thereby enhancing the proportion of expression of the α 1L -AR phenotype. The α 1L -AR-expressing α 1a -COE( α 1L -dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of α 1L -AR-targeted drugs.

Phenotype pharmacology of lower urinary tract α1‐adrenoceptors

“…More recently, this conclusion was again confirmed in a recombinant system (Nishimune et al, 2010b). In the CHO cell line, transfection of a1a-adrenoceptor cDNA predominantly expresses a1A-adrenoceptor phenotype, with an extremely minor proportion of a1L-adrenoceptor (less than 10% of total a1-adrenoceptor).…”

Influence of Tissue Integrity on Pharmacological Phenotypes of Muscarinic Acetylcholine Receptors in the Rat Cerebral Cortex

“…Cells were harvested by gentle scraping without using trypsin. The whole-cell binding assay was essentially the same as that of the homogenate binding experiment described above and previously (Nishimune et al, 2010). Incubation was carried out at 4°C for 4 h.…”

“…The pharmacological and biochemical properties of G protein-coupled receptors, including mAChRs, were demonstrated to be more complex than originally supposed (Christopoulos and Kenakin, 2002;van Koppen and Kaiser, 2003;Bockaert et al, 2004;Baker and Hill, 2007;Ramachandran et al, 2009;Nishimune et al, 2010). Most G protein-coupled receptors possess not only orthosteric binding sites but additional binding sites such as allosteric sites, form multiple conformations, and activate different signaling pathways.…”

Discovery of potent α1L-adrenoceptor agonists: Design and synthesis of bicyclic derivatives

“…Putative α 1L -adrenoceptors had been discussed for several years and recent research results have confirmed the existence of α 1L -adrenoceptor subtypes. Muramatsu and co-workers have reported an α 1L -adrenoceptor-expressing cell line 3 . The α 1L -adrenoceptor mediate contraction of the rabbit prostate 4 , the mouse prostate 5,6 , the rat urethra 7 , the human bladder 7 and prostate 8 , and other mammalian urogenital structures.…”

“…We evaluated substituent effects for MK017 and discovered the 3-isopropyl substituent is essential for α 1L -adrenoceptor agonist activity (Table 1). Agonist activity is indicated as the ratio of noradrenaline (NA) half maximal effective concentration (EC 50 ) in Chinese hamster ovary (CHO) cells stably expressing human α 1L -adrenoceptor 3,10,11 . We subsequently hypothesized that conformational restricted compounds in which the 2′ carbon was connected to the 1′, 3′, or 3′′ carbon via a C1 or C2 unit, while retaining the isopropyl unit, would achieve high α 1L -adrenoceptor agonist activity.…”