G Protein-Coupled Receptors in the Sweet Spot: Glycosylation and other Post-translational Modifications

Goth, Christoffer K.; Petäjä-Repo, Ulla E.

doi:10.1021/acsptsci.0c00016

Cited by 52 publications

(57 citation statements)

References 92 publications

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“…Depending upon the receptor conformation stabilized by the ligand, multiple distinct pathways can be activated or repressed. To maintain their exquisite spatial and temporal specificity, GPCRs are tightly regulated by post-translational modifications that affect their trafficking to the cell surface, internalization and down-regulation (5).…”

Section: Introductionmentioning

confidence: 99%

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

Coleman

Ngo

Smythe

et al. 2020

Preprint

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GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn 105 . The smaller species is produced by matrix metalloprotease/ADAMmediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the Nterminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal 'tethered' counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

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Section: Introductionmentioning

confidence: 99%

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

Coleman

Ngo

Smythe

et al. 2020

Preprint

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“…It is not clear why the two forms of the receptor exist in adipocytes or other cell types. It has been suggested that the glycosylation state of G protein-coupled receptors (GPCR) could influence transport to the plasma membrane [ 46 ]), ligand binding and affinity [ 33 ], degradation [ 47 ], and the formation of heterodimers with other GPCRs [ 48 – 50 ]. Clearly, further studies are needed to understand the functional significance of the two OXTR isoforms.…”

Section: Discussionmentioning

confidence: 99%

Oxytocin reduces adipose tissue inflammation in obese mice

et al. 2020

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Background: Obesity and adipose tissue expansion is characterized by a chronic state of systemic inflammation that contributes to disease. The neuropeptide, oxytocin, working through its receptor has been shown to attenuate inflammation in sepsis, wound healing, and cardiovascular disease. The current study examined the effects of chronic oxytocin infusions on adipose tissue inflammation in a murine model of obesity, the leptin receptordeficient (db/db) mouse. Methods: The effect of obesity on oxytocin receptor protein and mRNA expression in adipose tissue was evaluated by Western blotting and real-time polymerase chain reaction. Mice were implanted with osmotic minipumps filled with oxytocin or vehicle for 8 weeks. At study endpoint adipose tissue inflammation was assessed by measurement of cytokine and adipokine mRNA tissue levels, adipocyte size and macrophage infiltration via histopathology, and plasma levels of adiponectin and serum amyloid A as markers of systemic inflammation. Results: The expression of adipose tissue oxytocin receptor was increased in obese db/db mice compared to lean controls. In adipose tissue oxytocin infusion reduced adipocyte size, macrophage infiltration, IL-6 and TNFα mRNA expression, and increased the expression of the anti-inflammatory adipokine, adiponectin. In plasma, oxytocin infusion reduced the level of serum amyloid A, a marker of systemic inflammation, and increased circulating adiponectin. Conclusions: In an animal model of obesity and diabetes chronic oxytocin treatment led to a reduction in visceral adipose tissue inflammation and plasma markers of systemic inflammation, which may play a role in disease progression.

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“…The ability of E. coli to grow in media of defined composition facilitated labeling of recombinant CB 2 with stable isotopes 4 , 8 . However, E. coli cells lack the machinery for co- and post-translational modifications such as glycosylation and palmitoylation that are known to play a role in maintaining structural stability, cellular trafficking, and functional activity of GPCR in cell membranes 9 – 13 . Here, we explore the feasibility of mammalian cell-based platforms for expression of functional CB 2 receptor with native-like posttranslational modifications in large quantities.…”

Section: Introductionmentioning

confidence: 99%

“…1 H-13 C HSQC spectrum of methionine-13 C 5 -labeled CB 2 in Facade-TEG/phospholipid/CHS micelles recorded at 15 °C. The selected spectral region shows the sidechain resonances of 13 C-labeled methionine.…”

mentioning

confidence: 99%

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Yeliseev

Berg

Zoubak

et al. 2020

Sci Rep

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Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E.coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

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G Protein-Coupled Receptors in the Sweet Spot: Glycosylation and other Post-translational Modifications

Cited by 52 publications

References 92 publications

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

Oxytocin reduces adipose tissue inflammation in obese mice

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

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