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

Two distinct α1-adrenoceptor phenotypes (α1A- and α1L-ARs) are known to originate from a single ADRA1A(α1a) gene by an as-yet-unknown mechanism. We hypothesized that an α1a-AR-interacting protein could generate the α1L-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of α1A and α1L phenotypes. Cysteine-rich epidermal growth factor-like domain 1α (CRELD1α) was first identified using a yeast two-hybrid approach as an α1a-AR-interacting protein. Transfection of α1a-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing α1A-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (α1L-AR). Knockdown of endogenous CHO-CRELD1α [α1a-CKD(α1A-enhanced) cells] enhanced the expression of α1A-AR, whereas over-expression of CRELD1α reduced α1A-AR expression, yielding α1a-COE(α1L-dominant) cells expressing a high proportion (50%) of the α1L-AR phenotype. The ligand binding and functional agonist and antagonist profiles in α1a-CKD(α1A-enhanced) and α1a-COE(α1L-dominant) cell lines were entirely in accord with the α1A-AR and α1L-AR phenotypes observed in intact tissues. CRELD1αdown-regulates expression of the α1A-AR, thereby enhancing the proportion of expression of the α1L-AR phenotype. The α1L-AR-expressing α1a-COE(α1L-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of α1L-AR-targeted drugs. Keywords:: cysteine-rich epidermal growth factor–like domain 1α (CRELD1 α), α1L-adrenoceptor, α1A-adrenoceptor, phenotype pharmacology