Identification of Cysteine-Rich Epidermal Growth Factor–Like Domain 1α (CRELD1α) as a Novel α_1A-Adrenoceptor–Down-Regulating Protein and Establishment of an α_1L-Adrenoceptor–Expressing Cell Line

Abstract

Two distinct α₁-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 1α (CRELD1α) 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-CRELD1α [α_1a-CKD(α_1A-enhanced) cells] enhanced the expression of α_1A-AR, whereas over-expression of CRELD1α 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. CRELD1α 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.