BMPR1A Human, IgG-His

What is the molecular structure and function of BMPR1A?

BMPR1A is a type I receptor in the bone morphogenetic protein (BMP) receptor family that forms heterodimers with type II BMP receptors upon ligand binding. The receptor contains an extracellular ligand-binding domain, a transmembrane region, and an intracellular kinase domain. When activated, BMPR1A phosphorylates Smad1/5/8 proteins, which subsequently form complexes with Smad4 and translocate to the nucleus to regulate target gene expression . BMPR1A is the predominant receptor during specific developmental periods, including oligodendroglial and interneuron generation . The receptor plays essential roles in multiple stem cell populations, regulating differentiation and self-renewal processes.

Which BMP ligands preferentially bind to BMPR1A?

BMPR1A can bind multiple BMP ligands, with varying affinities. Research indicates interactions with BMP2, BMP4, BMP6, BMP7, and BMP9, though with different binding affinities and downstream signaling consequences . These interactions trigger the formation of receptor complexes that activate the canonical Smad1/5/8 pathway and can also initiate non-canonical signaling through MAPK pathways, including p38, ERK, and JNK cascades. The specific downstream effects depend on the particular ligand-receptor combination and the cellular context.

How does BMPR1A signaling differ from other BMP receptors?

BMPR1A has unique functions compared to other type I BMP receptors such as BMPR1B and ACVR1 (ALK2). Studies using conditional knockout models demonstrate that these receptors cannot fully compensate for each other . In neural development, BMPR1A specifically regulates oligodendrocyte and interneuron numbers . Similarly, in immune responses, BMPR1A has distinct roles in germinal center dynamics and establishment of long-lived humoral immunity . These specific functions reflect differential expression patterns, ligand binding preferences, and downstream signaling capabilities that distinguish BMPR1A from other BMP receptors.

What are the optimal conditions for using recombinant BMPR1A-IgG-His in binding assays?

For optimal binding assay performance with BMPR1A-IgG-His, researchers should consider several parameters:

• Buffer composition: PBS (pH 7.2-7.4) with 0.1% BSA provides stable conditions

• Temperature: Perform binding assays at 25°C for routine applications or 4°C for extended incubations

• Incubation time: 1-2 hours is generally sufficient for equilibrium binding

• Washing steps: Use PBS with 0.05% Tween-20 for reduced background

• Detection methods: Anti-His or anti-IgG antibodies conjugated to HRP, fluorophores, or biotin

When comparing binding affinities of different BMP ligands, standardized competitive binding assays with gradient concentrations (0.1-100 nM) are recommended. The dual tag system allows for flexible immobilization strategies - either capture via the His-tag on Ni-NTA surfaces or via the IgG portion on protein A/G plates.

How can I establish BMPR1A knockout or conditional knockout models for functional studies?

Based on the literature, several successful approaches have been used to generate BMPR1A knockout models:

• Germline deletion: Complete BMPR1A knockout is embryonically lethal at gastrulation stage , necessitating conditional approaches for most studies

• Conditional knockout using Cre-loxP system:

  • Generate or obtain mice with floxed BMPR1A alleles (BMPR1A^fx/fx)

  • Cross with tissue-specific Cre lines (e.g., P0-Cre for neural crest, Olig1-Cre for oligodendroglial lineage)

  • Confirm deletion efficiency through PCR detection of deleted exon 2

• Cell-specific targeting approaches:

  • For B-cell specific deletion, CD19-Cre or MB1-Cre systems have been used

  • For mammary tissue studies, MMTV-Cre systems have been employed

Verification of knockout should include qPCR analysis for BMPR1A transcript levels (targeting exon 2), western blot for protein expression, and functional assays showing disrupted BMP-Smad1/5/8 signaling. Reporter systems using BMPR1A.IRES.EGFP constructs can also be valuable for tracking expression patterns .

What methodologies are recommended for analyzing BMPR1A-mediated signaling pathways?

To comprehensively analyze BMPR1A-mediated signaling, researchers should employ multiple complementary approaches:

• Phosphorylation dynamics:

  • Western blotting for pSmad1/5/8 levels at multiple time points (5, 15, 30, 60 minutes) following ligand stimulation

  • Analysis of non-canonical pathway activation: p38 MAPK, ERK, JNK phosphorylation states

• Transcriptional regulation:

  • qRT-PCR for direct Smad target genes (ID1, ID2, ID3, ID4)

  • Genome-wide approaches like RNA-seq to identify broader transcriptional effects

  • Chromatin immunoprecipitation (ChIP) to map Smad binding sites

• Protein-protein interactions:

  • Co-immunoprecipitation to detect BMPR1A associations with type II receptors and downstream effectors

  • Proximity ligation assays for visualizing interactions in situ

• Functional readouts:

  • Cell-type specific differentiation markers (e.g., adipogenic markers PPARγ, FABP4, and adiponectin in MSC studies)

  • Lineage tracing in developmental studies

  • Flow cytometry for immune cell population analysis in germinal center studies

How does BMPR1A function in germinal center dynamics and humoral immunity?

BMPR1A plays crucial roles in germinal center (GC) B cell responses and the establishment of long-lived humoral immunity. Expression studies using qPCR and BMPR1A.IRES.EGFP reporter mice have demonstrated that BMPR1A expression is upregulated in GC B cells (GCBC) and subsets of memory B cells (MBC), bone marrow plasmablasts, and bone marrow plasma cells (BMPC) .

In mice with B cell-targeted BMPR1A deletions, researchers observed:

• Initially diminished GC response

• Subsequent recovery of GCBC compartment size, with accumulation of cells carrying unmodified rather than deleted BMPR1A alleles

• Strong selective pressure for retention of functional BMPR1A alleles

• Permanent marked reduction in:

  • Switched bone marrow antibody-forming cells

  • Bone marrow plasma cells

  • Memory B cells

  • Antigen-specific serum IgM

These findings indicate that BMPR1A signaling is critical for the modulation of B cell responses and the establishment of long-term immunological memory. The selective retention of functional BMPR1A in responding cells underscores its essential role in these processes.

What role does BMPR1A play in oligodendrocyte and interneuron development?

BMPR1A signaling critically determines the numbers of oligodendrocytes and calbindin-positive interneurons during neural development. Research using conditional BMPR1A knockout in Olig1-expressing progenitors has revealed:

• Developmental effects:

  • At birth: Fewer immature oligodendrocytes in BMPR1A-null brains

  • By postnatal day 20: Significantly higher numbers of both immature and mature oligodendrocytes compared to wild-type mice

  • Significant increase in calbindin-expressing interneurons in the dorsomedial cortex at birth

• Cellular mechanisms:

  • Decreased cell cycle length in subventricular zone progenitor cells

  • Altered expression of lineage-specific transcription factors

  • BMPR1A mediates the suppressive effects of BMP signaling on oligodendroglial and interneuron lineage commitment

The whole brains of conditional knockout mice showed approximately 80% reduction in mRNA encoding intact BMPR1A receptor compared with wild-type brains, suggesting that Olig1-positive progenitors constitute a large proportion of the BMPR1A-expressing cells in the developing brain .

How does BMPR1A signaling influence adipogenic differentiation of mesenchymal stem cells?

BMPR1A plays a pivotal role in directing mesenchymal stem cell (MSC) differentiation toward adipogenic lineages. Research on MSCs from ankylosing spondylitis (AS) patients demonstrated:

• Increased BMPR1A expression in ASMSCs compared to healthy donor MSCs (HDMSCs)

• Enhanced adipogenic differentiation capacity in ASMSCs

• Activation of the BMP-pSmad1/5/8 signaling pathway during adipogenesis

• Silencing BMPR1A using shRNA eliminated the difference in adipogenic differentiation between HDMSCs and ASMSCs

The mechanism involves:

• Binding of BMPs to BMPR1A to form heterodimers

• Phosphorylation of Smad1/5/8

• Activation of the BMP-pSmad1/5/8 signaling pathway

• Upregulation of adipogenic markers (PPARγ, FABP4, and adiponectin)

• Increased fat content in bone marrow adjacent to affected joints

These findings suggest that BMPR1A-mediated signaling is a key determinant of MSC fate decisions, with increased expression potentially contributing to pathological fat metaplasia and abnormal bone formation in certain disease states.

What are common pitfalls when working with BMPR1A constructs in cell culture systems?

Researchers should be aware of several technical challenges when working with BMPR1A constructs:

• Expression level variability:

  • Endogenous BMPR1A expression varies significantly between cell types

  • Transfection efficiency can affect interpretation of overexpression studies

  • Recommended: Quantify endogenous BMPR1A levels by qPCR and western blot before experimental design

• Receptor complex formation issues:

  • BMPR1A requires type II receptors for signaling

  • Cell lines may have limiting amounts of type II receptors

  • Solution: Co-transfect with appropriate type II receptors when necessary

• Tag interference concerns:

  • IgG and His tags may partially interfere with ligand binding or receptor complex formation

  • Control experiments comparing tagged and untagged constructs are advised

  • The relative position of tags (N-terminal vs C-terminal) can affect functionality

• Ligand selection considerations:

  • Different BMP ligands have varying affinities for BMPR1A

  • Commercial recombinant BMPs may vary in bioactivity between lots

  • Multiple ligand concentrations should be tested (typically 5-100 ng/ml range)

• Signaling detection windows:

  • Smad1/5/8 phosphorylation peaks at different times depending on cell type (typically 15-60 minutes)

  • Perform time-course experiments to identify optimal sampling points

How can I distinguish between canonical and non-canonical signaling downstream of BMPR1A?

Distinguishing between canonical (Smad-dependent) and non-canonical (Smad-independent) signaling downstream of BMPR1A requires systematic analytical approaches:

• Temporal analysis:

  • Canonical Smad1/5/8 phosphorylation typically occurs rapidly (within 5-30 minutes)

  • Non-canonical pathway activation (p38, ERK, JNK) often follows different kinetics

  • Conduct detailed time-course experiments (5, 15, 30, 60, 120 minutes post-stimulation)

• Pathway-specific inhibitors:

  • Dorsomorphin or LDN-193189 to inhibit BMPR1A kinase activity

  • SB203580 for p38 MAPK inhibition

  • PD98059 for ERK pathway inhibition

  • SP600125 for JNK inhibition

• Genetic approaches:

  • Smad1/5 knockdown or knockout to eliminate canonical signaling

  • Constitutively active BMPR1A (caALK3) to study pathway selectivity

  • Dominant-negative Smad constructs

• Readout selection:

  • ID1/2/3/4 gene expression as canonical pathway reporters

  • Pathway-specific transcription factor activation (e.g., ATF2 for p38 pathway)

  • Phospho-specific antibody arrays for broader pathway screening

• Single-cell analysis:

  • Immunofluorescence to detect heterogeneity in pathway activation

  • Flow cytometry for quantitative assessment of signaling in cell populations

What are the consequences of BMPR1A mutations in human disease?

BMPR1A mutations are associated with several human pathologies:

• Juvenile Polyposis Syndrome (JPS):

  • Autosomal dominant condition caused by heterozygous truncating variants or deletions

  • Characterized by multiple hamartomatous polyps in gastrointestinal tract

  • Increased risk of colorectal cancer

• Congenital heart defects:

  • Heterozygous deletions associated with cardiac anomalies

  • Populations with atrioventricular septal defects show enrichment for rare missense BMPR1A variants

• Developmental disorders:

  • A homozygous missense variant in BMPR1A has been reported to cause a distinct clinical phenotype including:

    • Skeletal abnormalities

    • Growth failure

    • Large atrial septal defect

    • Severe subglottic stenosis

    • Laryngomalacia

    • Facial dysmorphisms

    • Developmental delays

• Ankylosing Spondylitis:

  • Increased BMPR1A expression in mesenchymal stem cells

  • Enhanced adipogenic differentiation

  • Contribution to fat metaplasia and new bone formation

The functional consequences of BMPR1A mutations include altered phosphorylation of Smad1/5/8, disrupted Sox9 expression via decreased phosphorylation of p38, and increased chondrocyte death in cells with mutated receptors .

How might targeting BMPR1A be useful in therapeutic development?

Based on research findings, BMPR1A represents a promising therapeutic target for several conditions:

• Cancer therapeutics:

  • BMPR1A deletion impairs mammary tumor formation

  • Targeting BMPR1A may inhibit tumor growth and progression

  • Potential approach: small molecule inhibitors of BMPR1A kinase activity

• Immunomodulatory applications:

  • BMPR1A regulates germinal centers and long-lived humoral immunity

  • Potential for modulating antibody responses in autoimmune diseases

  • Possible approach: antibodies targeting specific BMPR1A epitopes to modify signaling

• Neurological disorders:

  • BMPR1A signaling determines oligodendrocyte and interneuron numbers

  • Therapeutic potential in demyelinating diseases and neurodevelopmental disorders

  • Targeted delivery approaches needed for CNS applications

• Bone and cartilage diseases:

  • Modulation of BMPR1A signaling could influence MSC differentiation pathways

  • Potential applications in ankylosing spondylitis and other spondyloarthropathies

  • Combinatorial approaches targeting both BMPR1A and downstream effectors

Therapeutic strategies must consider the complex, context-dependent roles of BMPR1A in different tissues and developmental stages. Given its fundamental roles in multiple biological processes, targeted or tissue-specific approaches would be necessary to avoid unintended consequences of systemic modulation.

What are emerging areas of BMPR1A research with significant potential?

Several cutting-edge research areas involving BMPR1A show promise for significant advances:

• Single-cell transcriptomic analyses:

  • Characterizing BMPR1A expression and signaling at single-cell resolution

  • Identifying previously unrecognized cell populations dependent on BMPR1A

  • Understanding cellular heterogeneity in response to BMP ligands

• Structural biology approaches:

  • Cryo-EM studies of BMPR1A-containing receptor complexes

  • Structure-based design of selective BMPR1A modulators

  • Understanding the structural basis for ligand discrimination

• Tissue engineering applications:

  • Controlling stem cell fate through precise modulation of BMPR1A signaling

  • Development of biomaterials incorporating BMP ligands with controlled release

  • Engineering three-dimensional tissues with spatially regulated BMPR1A activation

• Immune system regulation:

  • Further characterization of BMPR1A roles in various immune cell populations

  • Exploration of BMPR1A as a target for vaccine adjuvants to enhance humoral immunity

  • Investigation of BMPR1A signaling in trained immunity and immune memory

• Precision medicine approaches:

  • Identification of patient-specific BMPR1A variants and their functional consequences

  • Development of tailored therapeutic strategies based on individual BMPR1A signaling profiles

  • Biomarker development for predicting response to BMP pathway-targeting therapies