Prolactin Human, His

What is the structural composition of human prolactin and how does it differ from other mammalian species?

Human prolactin is a single-chain polypeptide hormone consisting of 200 amino acids with a molecular mass of approximately 23 kDa. The amino acid sequence, as determined through recombinant techniques, is: MLPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCH TSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEI EEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDS HKIDNYLKLLKCRIIHNNNC .

Unlike prolactin from other species, human prolactin shares significant structural homology with human growth hormone, which historically complicated its identification and isolation. This similarity led many researchers before 1970 to doubt the existence of a distinct human prolactin hormone separate from growth hormone . The structural similarities are substantial enough that immunological tools specifically designed to distinguish between the two hormones were necessary for the ultimate confirmation of human prolactin as a separate entity .

Researchers should note that while the primary structure of prolactin is conserved across mammalian species, subtle differences exist in glycosylation patterns and post-translational modifications that may affect experimental approaches when working with cross-species models.

How is human prolactin synthesized and regulated in the body?

Human prolactin synthesis occurs primarily in lactotroph cells of the anterior pituitary gland, though extrapituitary synthesis also takes place in several tissues. The regulation of prolactin synthesis involves complex neuroendocrine pathways:

• Hypothalamic Regulation: Prolactin immunoreactivity is found within numerous hypothalamic areas across various mammals, including the dorsomedial, ventromedial, supraoptic, and paraventricular nuclei in rats . Evidence suggests that hypothalamic prolactin is synthesized locally, independent of pituitary production. Hypophysectomy studies indicate that removal of the pituitary gland has no effect on immunoreactive prolactin levels in the male hypothalamus and only partially reduces levels in female rat hypothalamus .

• Dopaminergic Inhibition: The primary regulatory mechanism for prolactin secretion is inhibitory control via dopamine released from hypothalamic neurons. This represents a unique regulatory paradigm among pituitary hormones, as most are primarily under stimulatory control.

• Transcriptional Control: The human prolactin gene expression is regulated by both proximal and distal promoter elements, with tissue-specific expression patterns being directed by alternative promoter usage.

When designing experiments to study prolactin regulation, researchers should consider that prolactin-secreting cell lines like GH cell lines differ from primary pituitary cells in important ways. Unlike primary lactotrophs, GH cell lines rapidly release rather than store synthesized prolactin and lack functional dopamine receptors, making them resistant to dopamine's inhibitory actions .

What are the principal physiological functions of prolactin beyond lactation?

While prolactin is primarily known for promoting breast milk production, research has identified numerous additional physiological roles:

• Reproductive Function: In males, prolactin likely enhances luteinizing hormone (LH) receptors in testicular Leydig cells, resulting in increased testosterone secretion when LH binds to these receptors, thus promoting spermatogenesis .

• Neurological Effects: Prolactin demonstrates neuroprotective effects on the central nervous system, promotes neurogenesis (formation of new neurons), and exhibits anti-stress and anxiolytic (anxiety-reducing) properties when present at physiologically normal levels .

• Immunomodulation: Prolactin plays functional roles in the immune system, influencing both innate and adaptive immune responses. The presence of prolactin receptors on multiple immune cell types suggests its importance in immune function regulation.

• Cellular Processes: The hormone is involved in fundamental cellular processes including growth, apoptosis, and differentiation across various tissues .

• Parental Behavior: Research suggests prolactin may have a role in preparing men for parental behavior, increasing the desire to comfort their children .

These diverse functions highlight the importance of considering prolactin's pleiotropic effects when designing research studies, as interventions targeting prolactin may have widespread physiological consequences beyond the primarily investigated system.

What are the current methodologies for studying prolactin receptor signaling pathways?

Prolactin receptor (PRLR) signaling occurs primarily via JAK kinase pathways and can be studied through several methodological approaches:

• Cell-Based Assays: The NB2-11 cell proliferation assay represents a gold standard for measuring prolactin bioactivity, with biological activity typically measured as ED50 ≤ 1 ng/mL for high-quality recombinant human prolactin . This assay leverages the proliferative response of these cells to prolactin through receptor-mediated signaling.

• Phosphorylation Analysis: Western blotting for phosphorylated JAK2 and STAT5 provides direct evidence of prolactin receptor activation and downstream signaling cascade initiation. Researchers should optimize antibody selection for phospho-specific epitopes to maximize signal-to-noise ratios.

• Gene Expression Studies: Quantitative PCR measuring expression of prolactin-responsive genes provides indirect evidence of receptor activation and pathway functionality. Target genes should be validated in your specific cellular context through preliminary experiments.

• Receptor Binding Assays: Radioligand binding assays using I125-labeled prolactin can quantify receptor density and binding affinity, though these approaches are increasingly being replaced by fluorescence-based techniques using labeled prolactin variants.

• CRISPR-Cas9 Approaches: Modern gene editing techniques allow for precise modification of receptor components or downstream signaling molecules to elucidate pathway mechanics.

When designing signaling studies, researchers should note that cell lines often present different receptor expression patterns and signal transduction machinery compared to primary cells. For instance, GH cell lines lack functional dopamine receptors, which significantly alters their response to regulatory factors compared to primary lactotrophs .

How can researchers effectively differentiate between prolactin isoforms in experimental settings?

Human prolactin exists in multiple isoforms resulting from alternative splicing, proteolytic cleavage, and post-translational modifications. Differentiating between these isoforms requires specialized techniques:

When analyzing experimental samples, consider that certain physiological or pathological conditions may alter the distribution of prolactin isoforms. For example, pituitary adenomas may secrete distinct patterns of prolactin variants compared to normal tissue, potentially affecting bioactivity measurements .

What experimental approaches are most effective for investigating prolactin's role in non-lactational physiological functions?

To investigate prolactin's diverse physiological roles beyond lactation:

For neurological studies, researchers should consider that prolactin is found within numerous hypothalamic areas and maintains its presence even after hypophysectomy, suggesting local synthesis independent of pituitary production . This has important implications for experimental design when studying central nervous system effects.

What are the optimal procedures for reconstitution and storage of recombinant human prolactin?

Proper handling of recombinant human prolactin is crucial for maintaining its biological activity:

• Reconstitution Protocol:

  • Centrifuge the vial before opening to collect all material at the bottom

  • Gently pipette the recommended solution (typically sterile water at 0.1 mg/mL) down the sides of the vial

  • Avoid vortexing as this can denature the protein

  • Allow several minutes for complete reconstitution

• Storage Conditions:

  • For prolonged storage, dilute the reconstituted protein to working aliquots in a 0.1% BSA solution

  • Store at -80°C and avoid repeated freeze-thaw cycles which significantly reduce activity

  • Single-use aliquots are strongly recommended to preserve protein integrity

• Quality Control:

  • Verify protein purity via reducing and non-reducing SDS-PAGE (should be ≥95%)

  • Check endotoxin levels using Kinetic LAL method (should be ≤1 EU/μg)

  • Confirm biological activity through NB2-11 cell proliferation assay (ED50 should be ≤1 ng/mL)

The formulation of commercial recombinant human prolactin typically consists of a lyophilized preparation from a 0.2 μm filtered solution containing 10 mM sodium phosphate at pH 7.5 . Researchers should be aware that deviations from recommended handling procedures may significantly impact experimental outcomes.

How can researchers accurately measure prolactin levels in experimental and clinical samples?

Accurate measurement of prolactin levels requires careful consideration of methodology:

• Immunoassay Selection:

  • Enzyme-linked immunosorbent assays (ELISAs) provide high sensitivity and specificity

  • Radioimmunoassays (RIAs) remain the gold standard for some applications but present radioactivity concerns

  • Automated chemiluminescent immunoassays offer high throughput for clinical samples

• Sample Preparation:

  • Blood samples should be collected in standardized conditions, ideally in the morning, with the subject in a fasting state

  • Stress, exercise, and recent breast examination can artificially elevate prolactin levels

  • Samples should be promptly centrifuged and serum/plasma separated to prevent degradation

• Assay Validation:

  • Cross-reactivity with similar hormones (particularly growth hormone) should be assessed

  • Recovery experiments with spiked samples can verify assay accuracy

  • Serial dilution of high-concentration samples should yield linear results

• Macroprolactin Screening:

  • Polyethylene glycol precipitation can identify the presence of macroprolactin (prolactin bound to antibodies)

  • This high-molecular-weight form has reduced bioactivity but is detected by many assays, potentially leading to falsely elevated results

• Reference Standards:

  • Use internationally recognized standards for calibration

  • Consider that normal prolactin ranges differ between men (<15 ng/mL) and non-pregnant women (<25 ng/mL)

Researchers should note that certain medications can cause elevated prolactin levels, potentially confounding experimental results. Additionally, hypothyroidism, kidney disease, and chest injuries can increase prolactin levels independent of the experimental variable being studied .

What cell and animal models are most appropriate for studying human prolactin function?

Selection of appropriate experimental models is crucial for translational prolactin research:

• Cell Models:

  • GH cell lines (GH3, GH4C1) derived from pituitary tumors express both prolactin and growth hormone in varying ratios

  • Advantages include immortality, easy storage, and perpetual supply without animal sacrifice

  • Limitations include rapid release rather than storage of prolactin and lack of functional dopamine receptors

  • MCF-7 breast cancer cells express prolactin receptors and respond to prolactin stimulation

  • Primary lactotrophs offer more physiologically relevant responses but are more technically challenging to maintain

• Animal Models:

  • Rodent models are most common, though researchers should note species differences in prolactin regulation

  • Knockout models for prolactin or its receptor provide valuable insights into physiological functions

  • Hypophysectomized animals with selective hormone replacement allow isolation of prolactin effects

  • Transgenic models with tissue-specific prolactin or prolactin receptor expression can elucidate local actions

• Ex Vivo Systems:

  • Pituitary slices maintain cell-cell interactions while allowing for controlled experimental manipulation

  • Hypothalamic explants are valuable for studying central prolactin synthesis and action

When using cell lines rather than primary pituitary lactotrophs, researchers should recognize their dissimilarities. Unlike pituitary cells, prolactin synthesized by GH cell lines is rapidly released and not stored, with no intracellular degradation occurring . Additionally, most GH cells lack functional dopamine receptors, making them resistant to dopamine's inhibitory effects on prolactin secretion .

What experimental designs best elucidate the cross-talk between prolactin and other hormonal systems?

Investigating prolactin's interactions with other hormonal systems requires sophisticated experimental approaches:

• Co-stimulation Experiments:

  • Sequential or simultaneous administration of prolactin with other hormones can reveal synergistic or antagonistic effects

  • Time-course studies can elucidate the temporal dynamics of cross-talk mechanisms

  • Dose-response relationships often shift in the presence of interacting hormones

• Receptor Antagonist Studies:

  • Selective blockade of either prolactin receptors or receptors for interacting hormones can dissect pathway interactions

  • Combining antagonists at sub-maximal doses can reveal non-additive effects indicative of shared signaling components

• Signaling Pathway Analysis:

  • Phosphoproteomic approaches can identify shared phosphorylation targets between prolactin and other hormonal systems

  • RNA-seq analysis following combinatorial hormone treatments can reveal transcriptional convergence or divergence

  • Pharmacological inhibitors of specific signaling components can identify pathway nodes mediating cross-talk

• Conditional Genetic Models:

  • Tissue-specific deletion of prolactin receptors allows assessment of local interactions with other hormonal systems

  • Inducible receptor systems permit temporal control of receptor expression to study developmental aspects of cross-talk

Particularly important hormonal interactions to consider include the relationship between prolactin and luteinizing hormone in male reproductive physiology, where prolactin likely enhances LH receptors in testicular Leydig cells, resulting in increased testosterone secretion when LH binds to these receptors . Additionally, researchers should account for prolactin's interactions with the hypothalamic-pituitary-adrenal axis given its documented anti-stress and anxiolytic effects .

What are the key technical parameters for recombinant human prolactin use in research?

Researchers working with recombinant human prolactin should consider the following technical specifications:

Source: Technical specifications data adapted from commercial recombinant human prolactin documentation .

The amino acid sequence of human prolactin (200 amino acids) is: MLPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCH TSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEI EEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDS HKIDNYLKLLKCRIIHNNNC .

How do normal and abnormal prolactin levels manifest in research and clinical contexts?

Understanding reference ranges and clinical implications of prolactin levels is essential for research design and interpretation:

Elevated prolactin levels (hyperprolactinemia) can affect the body in various ways:

• In women: Ovarian dysfunction leading to menstrual problems and infertility

• In men: Testicular dysfunction resulting in decreased libido and erectile dysfunction

• In both sexes: Inappropriate lactation (galactorrhea) in non-pregnant, non-breastfeeding individuals

Common causes of hyperprolactinemia include:

• Prolactinoma: Benign pituitary tumor producing prolactin (most common cause)

• Medication effects: Certain antipsychotics, antidepressants, and antihypertensives

• Hypothyroidism (underactive thyroid)

• Kidney disease

• Chest wall injuries or stimulation

Researchers should note that prolactin testing alone cannot determine the cause of abnormal levels, but serves as a starting point for further diagnostic investigation .