Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules

Abstract

Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)—a key regulator of cardiovascular, urinary and CNS function—binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics

“…However, the functional implications of this proteolytic processing event remain unclear. We previously discovered the ADRA1D exists as a modular homodimer, with one ADRA1D protomer bound to the PDZ protein syntrophin and the dystrophin-associated protein complex, and the second ADRA1D promoter bound to the multi-PDZ domain scaffold scribble (32). Thus, we hypothesized NT cleavage represents a mandatory processing checkpoint during assembly of ADRA1D macromolecular complexes.…”

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics

“…In our previous analysis of ADRA1D, we observed weak activation by phenylephrine for both WT and ΔPDZ ADRA1D. However, overexpression of SCRIB and or syntrophins enhanced the DMR response, nearly 5 fold higher with SCRIB alone [13]. Furthermore, others have shown that DMR response profiles are cell type dependent [6], adding another layer of complexity to GPCR activation and function.…”

“…Once bound, PDZ-proteins may modulate GPCR pharmacodynamic properties via scaffolding effector proteins in close proximity, organizing GPCR complexes as discrete microdomains in cells, or linking GPCRs to non-canonical signaling events [2,3]. We previously demonstrated the Type I PDZ α 1D -adrenergic receptor (AR) forms a macromolecular complex with PDZ-proteins scribble (SCRIB) and multiple isoforms of syntrophin (SNTA, SNTB1, and SNTB2), which impart functionality and distinct cellular localization to the receptor [9–13]. The specific contributions of each PDZ protein for ADRA1D function and agonist efficacy in human cells was determined by DMR technology.…”

“…The specific contributions of each PDZ protein for ADRA1D function and agonist efficacy in human cells was determined by DMR technology. SCRIB and syntrophins bind C-terminal ADRA1D PDZ-ligands on discrete protomers within ADRA1D homodimers/oligomers, and in doing so, differentially regulate agonist efficacy [13]. …”

Label-Free Dynamic Mass Redistribution Reveals Low-Density, Prosurvival α1B-Adrenergic Receptors in Human SW480 Colon Carcinoma Cells

“…We recently showed that a 1 -AR subtypes associate with selective intracellular molecular scaffolds that dictate their pharmacodynamic and signaling characteristics (Lyssand et al, 2008(Lyssand et al, , 2010Camp et al, 2015). Interestingly, proteomic screening indicates cell-type-specific a 1 -AR:PDZ-protein complexes are formed in human SW480 colon cancer cells, suggesting a 1 -ARs may couple to noncanonical, G protein-independent signal transduction mechanisms in this cell line (Camp et al, 2015). Thus, we used label-free dynamic mass redistribution (DMR) assays to pharmacologically identify endogenous a 1 -AR subtypes functionally expressed in SW480 colon carcinoma cells, thereby facilitating their examination as potential antineoplastic drug targets.…”