PRL R Human

What is the Human Prolactin Receptor and what is its structural composition?

The Human Prolactin Receptor (PRL R) is a single chain membrane-bound protein belonging to the class 1 cytokine receptor superfamily. The receptor contains three distinct domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The PRL R mediates cellular activation by prolactin (PRL), a 23 kDa neuroendocrine pituitary hormone also known as lactotrophin, mamotrophin, or luteotropic hormone. The extracellular domain is responsible for ligand binding, while the intracellular domain initiates signaling cascades upon ligand binding .

Methodologically, researchers can study PRL R structure through:

• X-ray crystallography of the extracellular domain

• Site-directed mutagenesis to identify critical binding residues

• Computational modeling of receptor-ligand interactions

Where is PRL R expressed in human tissues and what techniques best detect this expression?

PRL receptors are found in numerous tissues throughout the human body, including:

PRL R expression can be detected through antibody-based techniques using specific antibodies like MAB1167 or AF1167, with optimal dilutions determined by each laboratory for specific applications .

What are the different isoforms of Human PRL R and how do they differ functionally?

Human PRL R exists in multiple isoforms resulting from transcriptional regulation and alternative splicing. While the extracellular domains of these isoforms are identical, their cytoplasmic domains vary in length and composition, leading to different signaling capabilities:

• Long form (LF): Contains the complete intracellular domain capable of activating multiple signaling pathways

• Short forms (SF): Result from alternative splicing of exon 11, can inhibit LF activity through heterodimerization

• Soluble form: Contains the 206 NH₂-terminal amino acids of the extracellular domain, is secreted by mammary epithelial cells and found in milk

The identification of exon 11 was particularly important as it led to discovery of novel short forms with relevance in physiological regulation and breast cancer. These different isoforms contribute to the tissue-specific responses to prolactin .

What are the key signaling pathways activated by PRL R and how are they regulated?

Upon binding of PRL to the receptor, several signaling cascades are initiated through a well-orchestrated process:

• PRL binding causes receptor dimerization

• Dimerization leads to binding and phosphorylation of Jak2

• Activated Jak2 phosphorylates STAT proteins and the long form of PRL R

• Additional pathways are activated including:

  • C-src and fyn kinases

  • Ras/Raf/MAP kinase pathway

The regulation of these pathways is complex and involves:

• Differential expression of receptor isoforms

• Formation of homo- and heterodimers even in the absence of PRL

• PRL acting as a conformational modifier of pre-formed dimers

• Intramolecular S-S bonds stabilizing receptor conformation

Experimentally, these pathways can be studied using phospho-specific antibodies, kinase activity assays, and gene reporter systems in appropriate cell models.

How does PRL R signaling in NK cells differ from other cell types and what are the immunological implications?

Human NK cell lines (NK-92 and YT cells) constitutively express PRL R, with NK-92 cells containing higher levels, which correlates with enhanced capacity to proliferate and lyse target cells in response to PRL stimulation. The interaction between PRL and cytokines in NK cells reveals important differences:

• PRL synergizes with IL-15 to improve NK cell proliferation in a dose-dependent manner without the double peak that manifests with IL-2

• In IL-2-stimulated NK cells, PRL enhances cytotoxicity by upregulating perforin gene expression without influencing FasL

• In IL-15-stimulated NK cells, PRL enhances cytotoxicity by upregulating both perforin and FasL gene expression, but not IFNγ

PRL increases expression of IL-2Rα on the membrane and IL-2 mRNA in cells, indicating that PRL upregulates NK cell function by improving positive feedback between IL-2 and IL-2R. Similar results are observed in the network between IL-15 and IL-15R .

These findings suggest a potential role for PRL in modulating anti-tumor immune responses through NK cell activation.

What mechanisms govern the inhibitory effects of short form PRL R on long form activity?

The inhibitory effect of short form (SF) PRL R on long form (LF) activity involves several molecular mechanisms:

• Heterodimerization: SF and LF form heterodimers even in the absence of PRL

• Conformational changes: PRL binding modifies the conformation of these heterodimers

• Signaling inhibition: SF/LF heterodimers show altered signaling capacity compared to LF/LF homodimers

This inhibitory mechanism has physiological importance in regulating PRL responses and may have implications in pathological conditions such as breast cancer. The ratio of SF to LF expression may therefore be a critical determinant of cellular responsiveness to PRL stimulation.

Researchers investigating this phenomenon should consider techniques such as co-immunoprecipitation, FRET analysis, and selective knockdown of specific isoforms to elucidate the molecular details of this regulatory mechanism.

What are the optimal methods for producing and purifying recombinant human PRL and PRL R for research?

Novel protocols have been developed for large-scale preparation of:

• Untagged human PRL (hPRL)

• hPRL antagonist (del 1-9-G129R hPRL)

• hPRL receptor extracellular domain (hPRLR-ECD)

The del 1-9-G129R hPRL antagonist prepared using newly described protocols shows similar biological properties to those prepared by original methods but with >6-fold improved yields . The interaction of this antagonist with hPRLR-ECD can be demonstrated through:

• Competitive non-radioactive binding assays using biotinylated hPRL

• Formation of stable 1:1 complexes under non-denaturing conditions

• Surface plasmon resonance methodology

For long-lasting hPRL needed for in vivo experiments, mono-pegylated analogues can be prepared, though researchers should note that pegylation may lower biological activity in homologous in vitro assays .

How can researchers develop improved PRL R antagonists for experimental applications?

To develop high-affinity PRL antagonists, researchers can employ yeast surface display methodology:

• Express del 1-9-G129R hPRL on the surface of yeast cells

• Verify retention of binding capacity for hPRLR-ECD

• Create a library of randomly mutated open reading frames of del 1-9-G129R hPRL

• Select high-affinity mutants through sequential rounds of enrichment

This methodology provides a platform for future development of high-affinity hPRL antagonists. The antagonist del 1-9-G129R hPRL inhibits hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR, making it a valuable tool for PRL R research .

For researchers developing antagonists, it's essential to validate their functionality through multiple assays including binding affinity measurements, cell proliferation assays, and signaling pathway analyses.

What cell-based models are most appropriate for studying PRL R function in different contexts?

Several cell models can be employed to study different aspects of PRL R biology:

When selecting a model system, researchers should consider:

• The specific PRL R isoform(s) expressed

• The downstream signaling pathways activated

• The functional readout most relevant to their research question

• The physiological relevance of the model system

How does PRL R contribute to breast cancer development and progression?

PRL R plays a complex role in breast cancer:

• PRL is a major hormone in the proliferation/differentiation of breast epithelium essential for lactation

• PRL is involved in breast cancer development, tumor growth, and chemoresistance

• The balance between long and short forms of PRL R may influence cancer progression

Research methodologies to investigate PRL R in breast cancer include:

• Analysis of receptor isoform expression in patient samples

• Correlation of expression patterns with clinical outcomes

• Functional studies using selective antagonists in cancer models

• Investigation of interactions between PRL R signaling and other oncogenic pathways

The development of specific PRL R antagonists may represent a potential therapeutic approach for certain breast cancer subtypes.

What experimental approaches best characterize the PRL-PRL R axis in immune modulation?

To study PRL R function in immune cells, researchers can employ:

• Flow cytometry to quantify receptor expression on specific immune cell populations

• Proliferation assays to assess functional responses to PRL stimulation

• Cytotoxicity assays to measure NK cell activity

• Gene expression analysis to identify regulated genes (perforin, FasL)

• Cytokine production assays to measure IL-2 and other immune mediators

When designing experiments, researchers should consider the synergistic effects between PRL and cytokines such as IL-2 and IL-15. The data indicates that PRL may enhance NK cell function through multiple mechanisms, including upregulation of cytolytic molecules and modulation of cytokine receptor expression .

These approaches allow for comprehensive characterization of how the PRL-PRL R axis contributes to normal immune function and potentially to immune-related disorders.

What are the emerging techniques for studying PRL R dynamics and trafficking?

Advanced imaging and molecular techniques are revolutionizing PRL R research:

• Live-cell imaging with fluorescently tagged receptors to track trafficking

• Single-molecule microscopy to study receptor dimerization kinetics

• CRISPR-Cas9 gene editing to create reporter cell lines

• Proximity labeling techniques to identify receptor-associated proteins

These approaches provide unprecedented insights into the temporal and spatial dynamics of PRL R signaling, which may reveal new regulatory mechanisms.

How can transcriptional regulation of PRL R be experimentally manipulated for research purposes?

Understanding and manipulating PRL R gene expression requires sophisticated approaches:

• Promoter analysis using reporter assays to identify regulatory elements

• ChIP-seq to map transcription factor binding sites at PRL R promoters

• Epigenetic profiling to characterize chromatin modifications

• Targeted gene activation/repression using CRISPR-based techniques

The human PRLR expression is controlled at the transcriptional level by multiple promoters, each directing transcription/expression of a specific non-coding exon 1, a common non-coding exon 2, and coding exons E3-11 . This complex regulatory architecture provides multiple points for experimental intervention to modulate receptor expression in research models.