Recombinant Human Protein GPR107 (GPR107)

What is GPR107 and what are its key structural characteristics?

GPR107 (also known as Lung seven transmembrane receptor 1) is an orphan G-protein-coupled receptor that contains multiple transmembrane domains. The protein undergoes post-translational processing, including cleavage by the endoprotease furin, with a disulfide bond connecting the two resulting fragments . The functional GPR107 protein has several conserved domains that are critical for its biological activity.

GPR107 is encoded by the GPR107 gene in humans, and there are three reported splice variants, though isoform 2 (UniProt accession number Q5VW38-2) appears to be the predominantly expressed form in many tissues . The complete protein contains 472 amino acids, though recombinant fragments are often used in research applications.

Where is GPR107 expressed in mammalian tissues?

GPR107 exhibits diverse tissue expression patterns with significant presence in:

• Central nervous system, particularly in the hypothalamus

• Cardiovascular tissues including heart

• Endocrine pancreas, especially in α-cells

• Gastrointestinal tissues, including expression in the KATOIII gastric tumor cell line

For experimental work, researchers have successfully detected GPR107 in KBM7 cells (a myeloid leukemia cell line), HeLa cells, HEK293T cells, and αTC1-9 cells (pancreatic α-cell line) .

How can researchers detect and quantify GPR107 expression in tissues and cells?

Detection of endogenous GPR107 presents challenges due to limited availability of high-quality antibodies. Researchers should consider the following methods:

mRNA detection:

• RT-PCR using primers specific to GPR107 gene (e.g., 5′-ATGGCCGCTCTGGCGCCCGTCGGCT-3′ and 5′-GGCCTTCTTGGTCATCAGTGC-3′)

• Normalizing to housekeeping genes such as GAPDH (for cell lines) or HPRT1 (for primary tissues like pancreatic islets)

Protein detection:

• Western blot analysis using commercially available GPR107 antibodies (e.g., Abcam, 1:500 dilution)

• For tagged recombinant proteins, anti-tag antibodies often provide better specificity than GPR107 antibodies

• Immunohistochemistry can be used to visualize co-localization with potential ligands or subcellular markers

What expression systems are used to produce recombinant GPR107?

Recombinant GPR107 can be produced using several expression systems:

E. coli expression system:

• Commonly used for producing protein fragments rather than full-length GPR107

• Typically yields protein with >90% purity as determined by SDS-PAGE

• Often includes affinity tags (such as N-terminal His-tag and C-terminal Myc-tag) to facilitate purification

Mammalian expression systems:

• Retroviral transduction methods have been used successfully to express GPR107 in mammalian cells

• KBM7 and HeLa cells stably expressing GPR107 constructs can be generated through retroviral infection followed by selection with G418 (0.8 mg/ml for HeLa and 1.2 mg/ml for GPR107 GT cells)

What is the role of GPR107 in bacterial toxin trafficking and intracellular transport?

GPR107 plays a critical role in the intracellular trafficking of bacterial toxins, particularly Pseudomonas aeruginosa exotoxin A (PE). A genome-wide haploid genetic screen in KBM7 cells identified GPR107 as an essential host factor for PE intoxication .

Cellular localization and transport function:

• GPR107 localizes predominantly to the trans-Golgi network (TGN)

• It is essential for retrograde protein transport, which is required for the trafficking of certain bacterial toxins from endosomes to the Golgi and ultimately to the endoplasmic reticulum

• The N-terminal region of GPR107 is particularly critical for its biological function in transport pathways

Experimental approaches for studying GPR107 in toxin trafficking:

• Haploid genetic screens in KBM7 cells with toxin selection

• CRISPR/Cas9-mediated gene editing to confirm phenotypes in different cell types

• Fluorescently labeled toxins to track intracellular movement in cells with normal vs. disrupted GPR107 expression

What evidence supports GPR107 as the receptor for neuronostatin?

Multiple lines of evidence suggest that GPR107 may function as the receptor for neuronostatin, a peptide hormone derived from the somatostatin preprohormone:

• Co-expression patterns: GPR107 is expressed in tissues that respond to neuronostatin, including hypothalamus, heart, pancreatic α-cells, and gastric cells

• Functional studies with knockdown approaches:

  • Knockdown of GPR107 using siRNA in cell lines abolishes cellular responses to neuronostatin

  • Rats injected with siRNA against GPR107 into the lateral cerebroventricle lost responsiveness to neuronostatin's effect on mean arterial pressure (MAP)

  • GPR107 siRNA-treated rats (2 μg/day for 2 days) showed blunted baroreflex sensitivity, indicating physiological relevance

• Specificity controls:

  • Knockdown of other orphan GPCRs like GPR56 did not affect neuronostatin responses

  • The effects were specific to neuronostatin signaling, as angiotensin II could still elevate MAP in GPR107 siRNA-treated rats

• Co-localization evidence:

  • Immunohistochemical studies have demonstrated co-localization of neuronostatin with GPR107 on target tissues

How does GPR107 participate in cellular signaling pathways?

GPR107 appears to be involved in multiple signaling pathways:

cAMP-independent PKA activation:

• Neuronostatin acts via GPR107 to increase cAMP-independent protein kinase A (PKA) signaling

• This pathway regulates proglucagon expression in pancreatic α-cells and may influence glucose metabolism

G-protein association:

• GPR107 may associate with G-proteins at the Golgi to regulate membrane transport processes

• The specific G-protein subtypes that interact with GPR107 remain to be fully characterized

Cardiovascular regulation:

• Central nervous system GPR107 activation influences mean arterial pressure and baroreflex sensitivity

• A 20% decrease in GPR107 mRNA levels in the brain was sufficient to reduce neuronostatin's central cardiovascular actions

What are effective methods for manipulating GPR107 expression in experimental systems?

RNA interference approaches:

• Cell culture siRNA:

  • Transfection of siRNA complexes targeting GPR107 (typically 100 nM) has achieved 43-76% knockdown efficiency in various cell types

  • In αTC1-9 cells, 76% reduction in GPR107 mRNA levels was sufficient to attenuate neuronostatin's actions

  • In primary rat islets, knockdown efficiencies of 43.7-56.2% have been reported

• In vivo siRNA administration:

  • Intracerebroventricular injection of GPR107 siRNA (2 μg/day for 2 days) effectively reduced GPR107 expression in rat brain regions

  • The effectiveness of knockdown should be verified by RT-PCR and, where possible, by Western blot analysis

Gene editing approaches:

• CRISPR/Cas9 gene editing has been successfully used to manipulate GPR107 expression, confirming findings from haploid genetic screens

Stable cell line generation:

• Retroviral transduction of GPR107 cDNA followed by antibiotic selection (G418) can generate stable overexpression lines

• Lentiviral delivery of shRNA against GPR107 with puromycin selection (1 μg/ml) for 1 week has been used to generate stable knockdown lines

What experimental techniques are used to study GPR107-ligand interactions?

While definitive receptor binding assays for GPR107 are still developing, several approaches have been used to investigate potential ligand interactions:

Functional assays:

• Gene expression analysis:

  • qRT-PCR to measure changes in target gene expression (e.g., proglucagon) following GPR107 activation or inhibition

  • Western blot analysis to measure changes in protein levels or phosphorylation states

• Physiological response measurements:

  • Blood pressure monitoring in rodents following central administration of neuronostatin, with or without GPR107 knockdown

  • Baroreflex sensitivity testing to assess cardiovascular regulatory function

Interaction analysis:

• Co-localization studies:

  • Dual and triple labeling immunohistochemistry to demonstrate spatial proximity of GPR107 and putative ligands like neuronostatin

• Receptor expression systems:

  • While not yet fully characterized for GPR107, expression of the receptor in null cell lines would allow for determination of receptor kinetics and binding properties

What are the important quality control parameters for recombinant GPR107 protein?

When working with recombinant GPR107 protein:

Purity assessment:

• SDS-PAGE analysis should confirm purity greater than 90%

• Mass spectrometry can verify protein identity and post-translational modifications

Functional verification:

• If using protein fragments, ensure they contain the relevant functional domains

• For the E. coli-expressed fragment (amino acids 40-263), confirmation that it contains the sequences necessary for your experimental application is essential

Storage and stability:

• Recombinant GPR107 is typically provided in Tris/PBS-based buffer with 5-50% glycerol for liquid formulations

• Lyophilized powder forms contain Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• For long-term storage, reconstitute to 0.1-1.0 mg/mL with 5-50% glycerol and store at -20°C/-80°C in aliquots

How can researchers overcome challenges in studying GPR107 function?

GPR107 research presents several challenges that can be addressed through careful experimental design:

Limited antibody availability:

• Use epitope-tagged versions of GPR107 (HA, Myc, or His tags) for detection when studying exogenously expressed protein

• Validate antibody specificity using GPR107 knockdown or knockout controls

Functional redundancy:

• Include appropriate controls when performing knockdown experiments (e.g., targeting other orphan GPCRs like GPR56 as negative controls)

• Consider graded knockdown approaches to establish dose-dependent relationships

Tissue-specific effects:

• When investigating GPR107 function in a new tissue or cell type, first confirm expression levels by RT-PCR

• Establish baseline responses to potential ligands before attempting knockdown experiments

What are the most promising research directions for GPR107?

Based on current findings, several research directions appear particularly promising:

Metabolic regulation:

• Further investigation of GPR107's role in pancreatic α-cells and glucose homeostasis

• Development of neuronostatin agonists and antagonists for potential therapeutic applications in metabolic disorders

Cardiovascular physiology:

• Deeper exploration of GPR107's involvement in baroreflex sensitivity and blood pressure regulation

• Investigation of tissue-specific GPR107 function using conditional knockout models

Intracellular trafficking:

• Elucidation of the complete mechanism by which GPR107 facilitates retrograde transport

• Identification of GPR107-interacting proteins in the trans-Golgi network

Drug delivery applications:

• Given its role in toxin trafficking, GPR107 may represent a target for improving intracellular delivery of therapeutic molecules

GPR107 Knockdown Efficiency in Different Experimental Systems

Common Expression Systems and Tags for Recombinant GPR107