Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands

Abstract

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) – key regulators of cardiovascular and central nervous system function – form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

N-glycosylation of α1D-adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis

“…1A are the results. In agreement with our previous studies 22,28 , the input lane demonstrates full length SNAP-α 1D is robustly expressed as a monomeric band at ~80 kDa, a larger, more intense band at ~90 kDa (Fig. 1A, arrow, Supplementary Fig.…”

“…2B, underlined). Furthermore, previously reported α 1D -AR interactors syntrophin 21,24,25 , members of the dystrophin-associated protein complex 23 , and scribble 22,25 were identified in the WT, but not NQQ samples (Supplementary Datas S1, S2). Together, these data provide compelling evidence that glycosylation of both N65 and N82 are necessary for proper biogenesis of full-length α 1D -AR, and disruption of these essential glycosylation sites results in early termination of α 1D -AR processing after TM2.…”

“…To test this possibility, HEK293 cells were transiently transfected with N-terminal SNAP-epitope tagged α 1D -AR cDNA constructs (SNAP-α 1D ). We have previously demonstrated the SNAP epitope-tag facilitates visual analysis of GPCR protein bands directly within polyacrylamide gels, and do not require nitrocellulose paper transfer or antibody staining, thus removing all potential false positive bands 22,28,50 . Incorporating this powerful technology, HEK293 cell lysates expressing SNAP-α 1D were lysed, denatured, and incubated with PNGase F, then subjected to polyacrylamide gel electrophoresis and near-infrared imaging (PAGE NIR).…”

“…at 37 °C according to manufacturer's instructions. After reaction was complete, samples were analyzed by PAGE NIR analysis using a LI-COR Odyssey CLx (LI-COR, Lincoln, NE) 22,28,50 Lentil lectin affinity purification. Cells were lysed in 20 mM Tris-HCl (pH 8), 200 mM NaCl, 5 mM DTT, and 1% NP-40 on ice for 20 min.…”

“…This was examined by quantifying WT, N65Q, N82Q and NQQ SNAP-α 1D plasma membrane expression levels in fixed HEK293 cells treated with the cell-impermeable SNAP substrate, BG-782 ( Fig. 3B,C) 22,28,50 . Cells were also treated with nuclear stain TO-PRO-3 to normalize for cell number.…”