Recombinant Mouse GRAM domain-containing protein 2 (Gramd2)

What is the domain structure of mouse GRAMD2?

Mouse GRAMD2 possesses a relatively simple domain structure compared to other GRAM family proteins. It contains an N-terminal unstructured region, a PH-like GRAM domain (which binds phosphoinositide lipids), and a C-terminal transmembrane domain (TMD) that anchors the protein to the ER membrane. Unlike GRAMD1 proteins, GRAMD2 lacks the sterol-binding VASt domain, suggesting functional specialization within the GRAM protein family .

How does GRAMD2 target to the plasma membrane?

GRAMD2 employs its GRAM domain to bind specifically to phosphoinositide lipids at the plasma membrane, particularly PI(4,5)P2 and PI(4)P. Biochemical studies using purified GRAMD2a cytosolic domain (amino acids 1-298) in liposome-binding assays demonstrated concentration-dependent binding to liposomes containing either PI(4)P or PI(4,5)P2, with apparent higher selectivity for PI(4,5)P2. This lipid-binding capacity is essential for GRAMD2's localization and function at ER-PM contact sites .

What cellular functions have been attributed to GRAMD2?

GRAMD2 functions primarily as an ER-anchored PM tether at specialized ER-PM contact sites. It has been strongly implicated in calcium signaling pathways, particularly in store-operated calcium entry (SOCE). Gene set enrichment analysis has shown that GRAMD2a expression positively correlates with calcium signaling pathways across human and mouse datasets. Additionally, GRAMD2 serves as a specific marker for AT1 cells in lung tissue, making it valuable for lung epithelial research .

What mouse models are available for studying GRAMD2 function?

Researchers have developed Gramd2-CreERT2;mTmG transgenic mice by crossing Gramd2-CreERT2 with mTmG reporter mice. Upon tamoxifen administration, these mice express GFP predominantly in AT1 cells, providing a valuable tool for studying AT1 cell biology in health and disease. Characterization studies have confirmed that GFP expression in these mice specifically labels subpopulations of AT1 cells (approximately 52.83 ± 3.42% of AQP5+ cells), with minimal labeling of other cell types, including AT2 cells (only 0.56 ± 0.56% of proSFTPC+ cells) .

What are the best methods for isolating GRAMD2-expressing cells from mouse lung tissue?

For isolating GRAMD2-expressing AT1 cells from Gramd2-CreERT2;mTmG mice, researchers have successfully employed fluorescence-activated cell sorting (FACS) to obtain GFP+ cells. Using a protocol that excludes endothelial and immune cells (CD31-CD34-CD45-), approximately 106,300 ± 11,077 GFP high cells per mouse can be isolated with >90% viability. Verification of AT1 identity can be performed using immunofluorescence staining for established AT1 markers including AQP5, PDPN, HOPX, and IGFBP2, which showed strong co-expression with GFP (98.43 ± 0.74% AQP5+, 79.42 ± 5.54% PDPN+, 75.61 ± 1.52% HOPX+, and 54.51 ± 3.86% IGFBP2+) .

How can researchers visualize GRAMD2 subcellular localization?

GRAMD2 localization can be visualized using fluorescently tagged constructs (such as GRAMD2a-eGFP or GRAMD2a-mCherry) in live-cell imaging experiments. These constructs reveal GRAMD2's characteristic pattern at discrete puncta along the cell cortex, representing ER-PM contact sites. Co-expression with ER markers (e.g., GFP-Sec61β) or PM markers can confirm its localization at these junctions. For endogenous GRAMD2 detection, immunofluorescence using specific antibodies against GRAMD2 can be employed, though this approach may require optimization for signal-to-noise ratio .

How are GRAMD2-marked ER-PM contact sites distinct from other contact sites?

GRAMD2-marked ER-PM contact sites represent functionally specialized domains that are physically distinct from other ER-PM contacts. Co-localization studies have shown that GRAMD2a-marked sites overlap significantly with E-Syt2/3-labeled contacts (established ER-PM tethers), while showing minimal overlap with GRAMD1a-marked sites. Specifically, only ~8% of GRAMD2a fluorescence pixels overlapped with GRAMD1a fluorescence pixels. This suggests that GRAMD2a and GRAMD1a mark functionally distinct populations of ER-PM contacts, which differ in their lipid dependencies and likely their physiological roles .

What happens to GRAMD2 localization during phosphoinositide depletion?

When plasma membrane PI(4,5)P2 is depleted through phospholipase C activation (using oxotermorine-M stimulation in cells expressing the muscarinic acetylcholine receptor), GRAMD2a rapidly dissociates from the PM and redistributes throughout the ER. Upon removal of the stimulus and recovery of PM PI(4,5)P2 levels, GRAMD2a returns to the PM, often with faster kinetics than PI(4,5)P2 biosensors, possibly due to its ability to bind PI(4)P in addition to PI(4,5)P2. This dynamic behavior contrasts with GRAMD1a, which remains associated with the cortex during PI(4,5)P2 depletion, confirming their distinct targeting mechanisms .

What experimental approaches can determine the lipid-binding specificity of GRAMD2?

The lipid-binding specificity of GRAMD2 can be rigorously determined through several complementary approaches:

• Liposome binding assays: Using purified recombinant GRAMD2 cytosolic domain (GRAMD2aΔTMD) with liposomes containing various phosphoinositides at different concentrations, followed by western blot analysis to quantify binding.

• Lipid strip assays: Employing nitrocellulose membranes spotted with different lipids to determine binding preferences of purified GRAMD2.

• Live-cell imaging with phosphoinositide manipulation: Using pharmacological tools (e.g., oxotermorine-M) or optogenetic approaches to deplete specific phosphoinositides while monitoring GRAMD2 localization.

• Structure-function analysis: Creating GRAMD2 mutants with alterations in the GRAM domain to identify specific residues critical for lipid binding .

Why is GRAMD2 considered a superior marker for AT1 cells compared to traditional markers?

GRAMD2 has emerged as a valuable AT1 cell marker due to its high specificity compared to traditional markers such as AQP5, PDPN, HOPX, and AGER, which lack complete specificity for AT1 cells in distal lung epithelium. Comprehensive characterization of Gramd2-CreERT2;mTmG mice has demonstrated that GFP expression driven by the Gramd2 promoter is predominantly restricted to AT1 cells, with minimal expression in AT2 cells (less than 0.5% overlap with proSFTPC). While GRAMD2 labeling captures approximately 53% of all AQP5+ AT1 cells, this specificity makes it particularly valuable for lineage tracing and functional studies of distinct AT1 cell subpopulations .

What is the significance of GRAMD2+ AT1 cell plasticity in lung research?

The plasticity of GRAMD2+ AT1 cells has significant implications for understanding lung regeneration and repair mechanisms. While AT2 cells have been well-established as progenitor cells in the distal lung, the potential contribution of AT1 cells to alveolar regeneration has been less clear, partly due to the lack of highly specific mouse models. The development of Gramd2-CreERT2;mTmG mice has provided a tool to specifically investigate AT1 cell fate and plasticity. Initial studies suggest that GRAMD2+ AT1 cells can form organoids in 3D culture conditions, indicating potential plasticity that may be relevant to lung regenerative processes in injury and disease contexts .

How can researchers isolate and culture GRAMD2+ AT1 cells for experimental studies?

A methodological approach for isolating and culturing GRAMD2+ AT1 cells includes:

• Isolation: Harvest lungs from tamoxifen-induced Gramd2-CreERT2;mTmG mice, digest with enzyme mixture (dispase/collagenase), and create single-cell suspensions.

• FACS purification: Sort for GFP high CD31-CD34-CD45- cells to isolate AT1 cells while excluding endothelial and immune cells.

• Culture conditions: For 3D culture studies, isolated cells can be embedded in Matrigel or similar extracellular matrix preparations, supplemented with appropriate growth factors.

• Verification: Confirm AT1 identity through immunostaining for multiple AT1 markers (AQP5, PDPN, HOPX, IGFBP2) and assess the absence of AT2 markers (proSFTPC).

• Functional assessment: Evaluate the capacity of these cells to form organoids or undergo phenotypic changes in different culture conditions or in response to various stimuli .

How does GRAMD2 contribute to store-operated calcium entry (SOCE)?

GRAMD2a appears to function as a pre-marker of ER-PM contact sites utilized for store-operated calcium entry (SOCE). When ER calcium is depleted (for example, using thapsigargin), STIM1 (an ER calcium sensor) relocates to these pre-existing GRAMD2a-marked ER-PM junctions. Expression of GRAMD2a results in expansion of cortical ER similar to that observed with STIM1 expression, supporting its role in organizing SOCE-specific contact sites. Gene set enrichment analysis confirms strong positive correlations between GRAMD2a and calcium signaling pathways across human and mouse datasets, while GRAMD1a does not show consistent correlation with calcium signaling .

What are the molecular mechanisms that differentiate GRAMD2 functions from GRAMD1?

The functional differentiation between GRAMD2 and GRAMD1 arises from their distinct domain architectures and targeting mechanisms:

• Domain structure: GRAMD2 possesses only a GRAM domain and transmembrane domain, while GRAMD1 proteins (GRAMD1a-c) also contain VASt domains involved in sterol transport.

• PM targeting mechanisms: GRAMD2 targets to the PM via its GRAM domain interaction with phosphoinositides (primarily PI(4,5)P2), whereas GRAMD1a targeting is phosphoinositide-independent, possibly involving a protein partner or other lipid species like cholesterol.

• ER-PM contact site specificity: These proteins mark physically distinct ER-PM contact sites with minimal overlap, suggesting they organize functionally specialized membrane contact domains.

• Cell-type specificity: GRAMD1a localization to ER-PM contacts is cell-type specific (present in Cos7, Hek293, HeLa, and Arpe19 cells but absent in U2OS or HCT116 cells), indicating differential regulation across cell types .

What are common pitfalls in recombinant GRAMD2 protein expression and purification?

While specific pitfalls for GRAMD2 purification aren't directly addressed in the provided materials, general challenges for membrane-associated proteins like GRAMD2 include:

• Solubility issues: The transmembrane domain can cause aggregation during expression and purification. Solution: Express only the cytosolic domain (e.g., amino acids 1-298, GRAMD2aΔTMD) with appropriate tags (6xHis) for soluble protein production.

• Protein stability: GRAM domain proteins may have stability issues. Solution: Optimize buffer conditions with appropriate pH, salt concentration, and consider adding glycerol or specific lipids to stabilize the protein.

• Functional verification: Ensuring the recombinant protein retains lipid-binding activity. Solution: Perform liposome binding assays with various phosphoinositides to verify function of purified protein, as demonstrated with GRAMD2aΔTMD .

How can researchers overcome challenges in visualizing GRAMD2-marked ER-PM contacts?

Visualization of GRAMD2-marked ER-PM contacts presents several technical challenges:

• Resolution limitations: Standard confocal microscopy may not fully resolve these nanoscale contacts. Solution: Employ super-resolution microscopy techniques such as STORM, PALM, or SIM for improved resolution of these structures.

• Dynamic nature: These contacts are dynamic and can change rapidly with cellular conditions. Solution: Use live-cell imaging with appropriate temporal resolution to capture these dynamics.

• Specificity of markers: Ensuring proper labeling of GRAMD2 vs. other ER-PM contact proteins. Solution: Use multiple markers simultaneously and employ appropriate controls, including GRAMD2 knockout cells.

• Quantification challenges: Accurately measuring the extent and distribution of contacts. Solution: Develop robust image analysis pipelines that can quantify parameters such as contact site number, size, and intensity .

What controls are essential when using Gramd2-CreERT2;mTmG mice for lineage tracing studies?

When using Gramd2-CreERT2;mTmG mice for lineage tracing, several critical controls should be implemented:

• Tamoxifen dose-response studies: Determine optimal tamoxifen dosing that provides specific labeling while minimizing potential toxicity or off-target effects.

• No-tamoxifen controls: Include mice that carry the transgene but receive no tamoxifen to assess potential leakiness of the Cre system.

• Time-course analysis: Examine GFP expression at various timepoints after tamoxifen administration to determine the stability and dynamics of labeling.

• Multi-marker validation: Verify the identity of GFP+ cells using panels of markers for AT1 cells (AQP5, PDPN, HOPX, IGFBP2) and exclude other cell types using negative markers (proSFTPC for AT2 cells).

• Single-cell transcriptomic validation: Perform scRNA-seq on GFP+ sorted cells to comprehensively characterize their molecular identity and heterogeneity .