G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by posttranslational modifications, including N-glycosylation of asparagine residues. α 1D-adrenergic receptors (α 1D-ARs)-key regulators of central and autonomic nervous system function-contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α 1D-AR between transmembrane domain 2 and 3. Labelfree dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α 1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α 1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α 1D-AR n-terminal glycosylation is required for complete translation of nascent, functional receptor. G protein-coupled receptors (GPCRs) are essential membrane proteins that regulate the vast majority of physiological functions in the human body. As a result, GPCRs have been estimated to be targeted by approximately one third of all currently approved medications 1. Adrenergic receptors (ARs) are a clinically relevant subfamily of GPCRs. Activated by the endogenous sympathetic neurotransmitters epinephrine and norepinephrine, adrenergic GPCRs consist of three major subtypes: α 1 , α 2 , and β. The α 1 sub-family-containing α 1A , α 1B , and α 1D subtypes 2-are targets for medications that regulate blood pressure 3,4 , bladder 5,6 , prostate 7,8 , and central nervous system function 9-11. Thus, understanding the molecular and cellular mechanisms regulating α 1-AR function will help spur the development of new medications associated with aberrant α 1-AR signaling, such as hypertension, PTSD, schizophrenia, and benign prostatic hypertrophy 12-15. Among the three α 1 subtypes, the α 1D-AR remains poorly understood due to technical challenges. Relative to the closely related α 1A and α 1B-AR subtypes, α 1D-AR displays limited functional responses and minimal plasma membrane expression when expressed in heterologous cell culture 16-18. Although pharmacologically detectable in intact isolated aortae in organ-tissue bath assays 19 , α 1D-AR functional expression rapidly disappears in primary vascular smooth muscle cell cultures within 24-48 hours 20. Also, immortalized cell lines that endogenously express α 1D-ARs have yet to be discovered. Combined, these experimental clues indicate the molecular and cellular ...
The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12DNAJ-PKAchepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
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