Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a

Li, Hao-Zheng; Shao, Xin; Shou, Li‐Li; Li, Ning; Liu, Yali; Xu, Zeng‐Guang; Guo, Zhan‐Yun

doi:10.1016/j.abb.2021.108872

Cited by 7 publications

(17 citation statements)

References 33 publications

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“…Although GHSR1a signals through the Gq pathway, it also activates the transcription factor CREB through kinases such as Ca 2+ /calmodulin kinase IV and protein kinase C [32]. Thus, a CRE-controlled luciferase reporter can be used to monitor GHSR1a activation, as demonstrated in previous studies [6,31,33].…”

Section: Resultsmentioning

confidence: 99%

“…Activation potency of the S -alkylated ghrelin analogs towards human GHSR1a was measured using a cAMP-response element (CRE)-controlled NanoLuc reporter according to our previous procedures [29,31]. Briefly, HEK293T cells were transiently cotransfected with an expression construct of human GHSR1a (pcDNA6/GHSR1a) and a CRE-controlled NanoLuc reporter vector (pNL1.2/CRE).…”

Section: Methodsmentioning

confidence: 99%

“…Binding activity of the S -alkylated ghrelin analogs with human GHSR1a was measured using the NanoBiT-based homogenous binding assay according to our previous procedures [29,31]. Briefly, human embryonic kidney (HEK) 293T cells were transiently transfected with the expression construct pcDNA6/sLgBiT-GHSR1a that encodes an N-terminally secretory large NanoLuc fragment (sLgBiT)-fused human GHSR1a.…”

Section: Methodsmentioning

confidence: 99%

“…3). We measured their binding activity with human GHSR1a using the NanoBiT-based homogenous binding assay [6,11,29,31], and measured their activation potency towards human GHSR1a using a CRE-controlled NanoLuc reporter [6,11,29,31]. Although GHSR1a signals through the Gq pathway, it also activates the transcription factor CREB through kinases such as Ca 2+ /calmodulin kinase IV and protein kinase C [32].…”

Section: Activity Of the S-alkylated Ghrelin Analogsmentioning

confidence: 99%

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Development of esterase-resistant and highly active ghrelin analogs via thiol-ene click chemistry

Shao

Shou

et al. 2022

Preprint

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The orexigenic peptide ghrelin exerts important functions in energy metabolism and cellular homeostasis by activating the growth hormone secretagogue receptor type 1a (GHSR1a), and thus has therapeutic potential to treat certain diseases. Native ghrelin carries an essential O-fatty acyl moiety at the side-chain of its third Ser residue; however, this posttranslational modification is susceptible to hydrolysis by certain esterases in circulation, representing a major route of in vivo inactivation of ghrelin. In the present study, we developed a novel approach to prepare various esterase-resistant ghrelin analogs via photo-induced thiol-ene click chemistry. A recombinant unacylated human ghrelin mutant carrying a unique Cys residue at the third position was reacted with commercially available end alkenes, thus various alkyl moieties were introduced to the side-chain of its unique Cys residue via a thioether bond. Among eleven S-alkylated ghrelin analogs, analog 11, generated by reacting with 2-methyl-1-octene, not only acquired much higher stability in human serum and fetal bovine serum, but also acquired moderately higher activity compared with native human ghrelin. Thus, the present study not only provided an efficient approach to prepare various esterase-resistant ghrelin analogs, but also produced a novel highly stable and highly active ghrelin analog with therapeutic potential.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Activity Of the S-alkylated Ghrelin Analogsmentioning

confidence: 99%

See 2 more Smart Citations

Development of esterase-resistant and highly active ghrelin analogs via thiol-ene click chemistry

Shao

Shou

et al. 2022

Preprint

Self Cite

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“…The NanoBiT-based homogenous binding assays were conducted according to our previous procedures [3,[16][17][18]. The expression constructs for the sLgBiT-fused GHSRs were transiently transfected into human embryonic kidney (HEK) 293T cells, preprint (which was not certified by peer review) is the author/funder.…”

Section: The Nanobit-based Homogenous Binding Assaysmentioning

confidence: 99%

LEAP2 is a more conserved ligand than ghrelin for fish GHSRs

Shao

Wang

et al. 2022

Preprint

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Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous competitive antagonist and an inverse agonist of the ghrelin receptor GHSR. However, its functions in lower vertebrates are not well understood. Our recent study demonstrated that both LEAP2 and ghrelin are functional towards a fish GHSR from Latimeria chalumnae, an extant coelacanth believed to be one of the closest ancestors of tetrapods. However, amino acid sequence alignment identified that the 6.58 position (Ballesteros-Weinstein numbering system) of most fish GHSRs are not occupied by an aromatic Phe residue, which is absolutely conserved in all known GHSRs from amphibians to mammals, and is responsible for human GHSR binding to its agonist, ghrelin. To test whether these unusual fish receptors are functional, we studied the ligand binding properties of three representative fish GHSRs, two from Danio rerio (zebrafish) and one from Larimichthys crocea (large yellow croaker). After overexpression in human embryonic kidney 293T cells, the three fish GHSRs retained normal binding to all tested LEAP2s, except for a second LEAP2 from L. crocea. However, they displayed almost no binding to all chemically synthesized n-octanoylated ghrelins, despite these ghrelins all retaining normal function towards human and coelacanth GHSRs. Thus, it seems that LEAP2 is a more conserved ligand than ghrelin towards fish GHSRs. Our results not only provided new insights into the interaction mechanism of GHSRs with LEAP2s and ghrelins, but also shed new light on the functions of LEAP2 and ghrelin in different fish species.

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The NanoBiT-Based Homogenous Ligand–Receptor Binding Assay

Liu

Guo

2022

Bioluminescence

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Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a

Cited by 7 publications

References 33 publications

Development of esterase-resistant and highly active ghrelin analogs via thiol-ene click chemistry

Development of esterase-resistant and highly active ghrelin analogs via thiol-ene click chemistry

LEAP2 is a more conserved ligand than ghrelin for fish GHSRs

The NanoBiT-Based Homogenous Ligand–Receptor Binding Assay

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