Protein tyrosine phosphatase receptors (PTPRs), a family of proteins encoded by 21 genes in humans, are signaling molecules composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The diverse extracellular domain shares homology with cell adhesion molecules and encompasses a wide range of physiological functions, the transmembrane domain makes them a type of membrane receptor protein and the intracellular domain contains either one or two highly conserved intracellular phosphatase domain (1, 2). Protein tyrosine phosphatase receptor type N (PTPRN, also known as IA-2 and ICA512) and PTPRN2 (also known as IA-2β and phogrin) are atypical members of this protein family. PTPRN consists of 979 amino acids encoded by the Ptprn gene, located on human chromosome 2q35, while PTPRN2 consists of 986 amino acids, and its Ptprn2 gene is located on human chromosome 7q36 (3).

Comparison of the amino acid sequences of human, mouse, and rat PTPRN revealed that the intracellular domain is over 97% conserved in these species, while the extracellular domain is 80–90% conserved. The intracellular domain of PTPRN2 is 92% conserved in human, mouse and rat, whereas the extracellular domain shows only 50–60% identity (3). PTPRN and PTPRN2 share 74% identity within the intracellular, but only 27% in the extracellular domains (4–6). A splice variant of human Ptprn lacking exon 13 encoding the transmembrane region (7, 8), as well as three splice transcripts of human Ptprn2 (9), have been detected in the pancreas. The extracellular domain of PTPRN and PTPRN2 proteins (hereinafter referred to as PTPRNs) show partial homology with the extracellular domain of regulated endocrine specific protein 18, which is another protein marker of neuroendocrine cells (10–12), indicating a potential physiological significance for their function (13).

Unlike other PTPR members, which have a tandem phosphatase domain, PTPRNs have a single phosphatase domain (34). Furthermore, the structure of the catalytic domain is altered in PTPRN and PTPRN2, suggesting that both proteins are pseudophosphatases. According to the bioinformatic definition, a pseudophosphatase is a member of phosphatase family that contains a mutation that predicts impairment or loss of its catalytic activity, regardless of whether this protein is enzymatically active or not (35). However, neither PTPRN nor PTPRN2 exhibit tyrosine phosphatase activity (36, 37). Furthermore, the enzymatic portion of PTPRNs can heterodimerize with another PTPRs and cause a 20% decrease in enzyme activity (38). However, the biological roles of PTPRNs have been investigated for several decades and have provided strong evidence that these proteins have acquired novel cellular roles beyond tyrosine dephosphorylation. In general, the sequence similarity between PTPRN and PTPRN2 suggests some levels of redundancy. Consequently, the effects on neuroendocrine cells are enhanced or observed only in double knockout (DKO) mice (39).

Loss of tyrosine phosphatase activity of PTPRNs due to mutation of two residues in the catalytic domain does not exclude the possibility that these proteins act as enzymes for other substrates. To date, no replacement substrate for PTPRN has been identified, but PTPRN2 has been reported to be able to dephosphorylate specific inositol phospholipids, including PI(3)P, PI(4,5)P2, but not PI(3,4,5)P3. When the transmembrane form of PTPRN2 was overexpressed in mammalian cells, it reduced plasma membrane PI(4,5)P2 levels in a dose-dependent manner (40). Other have reported that PTPRN2 and phospholipase C beta enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells. They also found that the expression of these genes was increased in these cells, which coincided with human metastatic relapse. The authors further suggested that depletion of PI(4,5)P2 by these enzymes releases the PI(4,5)P2‐binding protein cofilin into the cytoplasm where it increases cellular migration and metastatic capacity (41). However, we observed no significant changes in InsP3-dependent calcium oscillations in gonadotrophs from DKO mice, which argues against a physiologically significant reduction in phospholipase C activity (23).

Several lines of research with pancreatic β-cells have shown that the secretory pathway is affected by deletion of Ptprn and/or Ptprn2. These genes appear to be required to accumulate normal levels of insulin-containing vesicles and prevent their degradation (42). Global knockout of Ptprn led to impaired glucose-mediated insulin secretion (43), whereas overexpression of Ptprn in an insulinoma cell line led to increased insulin secretion (42). Ptprn2 knockout mice also show impaired glucose tolerance and reduced glucose-induced insulin secretion but was not sufficient to prevent the development of diabetes (27). However, the role of PTPRNs in exocytosis appears to be specific to β-cells. Renin release from dense core vesicles of neuroendocrine juxtaglomerular granular cells is not directly inhibited by DKO, but reflects reduced catecholamine release from sympathetic nerve endings (17). In female but not in male mice, it was originally suggested that DKO inhibits the accumulation and secretion of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone, leading to infertility (27). Subsequent studies have shown that this is not the case for these hormones, that the exocytotic pathway of other anterior pituitary cells is also intact, and that the levels of hormones secretes by melanotrophs from the intermediate pituitary lobe are higher in DKO animals (see below).

Immunocytochemistry performed on cultured cells suggests that PTPRN is colocalized with neurosecretory granules and it is not a resident plasma membrane protein (16). PTPRN2 has also been reported to be enriched in the membranes of β-cell secretory granules (44). Proteomics analysis also revealed the presence of PTPRN and PTPRN2, as well as peptidyl-glycine-α-amidating monooxygenase (PAM), a neuropeptide processing enzyme (45), in insulin secretory granules (46). PTPRN has also been identified in the secretory granules of chromaffin cells (47). Expression of a fusion construct between PTPRN2-enhanced green fluorescent protein in β-cells and pheochromocytoma PC12 cells revealed the presence of this chimera in dense-core secretory granules (48, 49). PTPRN has also been reported to tether insulin secretory granules to actin microfilaments via its association with the adapter protein syntrophin beta 2 (50, 51). The same group also reported that the F-actin modifier villin-1 regulates insulin granule dynamic and exocytosis downstream of PTPRN (52). However, PTPRNs were not detected in pituitary corticotroph dense core vesicles (53) and TT endocrine cells (54), unlike PAM, the typical enzyme for this organelle. This may provide a rationale for the lack of effects of DKO on pituitary corticotroph secretion. Furthermore, Sntb2 encoding syntrophin beta 2, and Vil1 encoding Villin-1, are unlikely to play this role in pituitary hormone release because these genes are not expressed or are below detection by single cell RNA sequencing in hormone-producing cells (30).

Solimena’s laboratory proposed the association of PTPRN with insulin secretory granules not only to explain the initiation of the exocytotic process, but also to have a post-exocytotic functions. First, they reported that exocytosis of secretory granules leads to insertion of PTPRN in the plasma membrane, which promotes calcium-dependent cleavage of its cytoplasmic domain by mu-calpain. It has been suggested that this cleavage results in the generation of a cytosolic fragment of PTPRN that is targeted to the nucleus, causing upregulation of insulin gene expression. Therefore, this new pathway links regulated exocytosis to the control of gene expression and suggests that calcium acts as a dual signal: it triggers exocytosis and activates the retrograde pathway (55). Second, the same group proposed signal transducer and activator of transcription 5 (STAT5) as the binding domain for the cytosolic fragment of PTPRN and described the synergy of glucose and growth hormone signaling (56). Third, the C-terminal fragment of PTPRN has been proposed to promote β-cell proliferation by linking signaling by STAT3 and STAT5 (57). Consistently with these findings, effects of DKO on gene expression and/or cell proliferation have also been observed in neuroendocrine cells of the hypothalamus and pituitary gland (see below). Furthermore, knockout of Ptprn has been reported to reduce, whereas overexpression of PTPRN increases proliferation and migration of glioma cells (58). PTPRN2 has also been suggested to play a role in other types of cancers (59).

Early work with the pituitary gland suggested that DKO directly affects gonadotroph functions, particularly in females but not in males. In parallel with the β-cell secretion model, it has been suggested that PTPRNs are required for LH secretion, and DKO causes a lack of LH surge and ovulation. Therefore, PTPRNs in female gonadotrophs have been described as crucial for the structure and function of the dense core secretory vesicles, implying sexual dimorphism in exocytotic LH release (27). However, proteomics analysis of dense core secretory vesicles was not performed in pituitary cells to elucidate the presence/absence of PTPRNs in dense core secretory vesicles in female/male gonadotrophs. In addition, the authors did not examine the status of hypothalamic neurosecretory neurons that control pituitary gonadotroph functions.

Pituitary gonadotroph gene expression and hormone secretion are controlled by GnRH-secreting neurons (60), whose function is critically dependent on connections with kisspeptinergic neurons (61). GnRH is released in a pulsatile manner in females and males, causing oscillatory release of LH, a secretory pattern required for gonadal spermatogenesis/oogenesis and steroidogenesis (62). Pulsatile GnRH/LH release is driven by “GnRH pulse generator”, a neuronal assembly in the arcuate nucleus of the hypothalamus (63), which consist of kisspeptin-secreting neurons that control the distal processing of GnRH neurons and their secretion at the median eminence (64). The pulsatile release of GnRH/LH is sufficient for male fertility, but female fertility also depends on the sustained release of GnRH called the surge, which is necessary for ovulation (65). A distinct population of kisspeptin neurons is located in the rostral periventricular region of the third ventricle (RP3V) and stimulates the cell bodies of GnRH neurons to release GnRH, which causes the LH surge necessary for ovulation (65). Both GnRH and kisspeptin neurons also express Ptprn+Ptprn2 (24, 25), so DKO may affect their functions.

In a recent study (23), we showed that the density of kisspeptin staining was significantly reduced in both the arcuate nucleus and the RP3V region of DKO mice ( Figures 1A, B ). Moreover, the expression of Gnrh1 and Kiss1 was decreased in the hypothalamic tissue of DKO animals ( Figure 1C ). Expression of the pituitary gonadotroph-specific genes Lhb, Fshb, and Gnrhr was also significantly reduced in females and males (23). These changes were accompanied by significantly reduced pituitary LH accumulation and released in both females and males, arguing against sexual dimorphism at the pituitary level ( Figure 1D ). Significant changes in ovarian steroidogenesis and gene expression were also observed in DKO females, leading to the delay in puberty and female reproductive organ development (23). Others have also reported a delay in onset of puberty in Ptprn2-only knockout females (22). Finally, DKO females were in constant diestrus, indicating a lack of ovulation, in contrast to control females that had a 4–5-day estrous cycle. However, no changes were observed in testicular steroidogenesis and spermatogenesis, and seminal vesicles development in DKO males (23). The interpretation of these findings is summarized in the scheme shown in Figure 1E .

A marker gene for the hypothalamic-pituitary-adrenal axis is Pomc, which is expressed in both the hypothalamus and the pituitary gland. In the hypothalamus, Pomc is expressed in the arcuate nucleus (66) and in the pituitary Pomc is expressed in corticotrophs and melanotrophs (67). DKO did not affect Pomc expression in the hypothalamus ( Figure 2A ), but dramatically increased expression in the pituitary ( Figure 2B ), although both tissues express PTPRNs. Pomc regulatory sequences in the pituitary and hypothalamic tissues differ (68), indicating that the tissue-specific PTPRNs actions are transcriptionally related. Single knockouts of Ptprn and Ptprn2 also increased Pomc expression, but of smaller amplitudes compared to DKO (69).

Pituitary expression of Pomc is stimulated by hypothalamic corticotropin-releasing hormone (68), which is a ligand for corticotropin-releasing hormone receptor 1 expressed in corticotrophs and melanotrophs (67). However, DKO did not affect Crh expression (69). Tbx19 is a common developmental transcription factor gene for corticotrophs and melanotrophs, whereas Pax7 is expressed only in melanotroph (67) and ts expression in the pituitary was significantly elevated in DKO females and males ( Figure 2C ). TBX19 controls terminal differentiation of both lineages and, in cooperation with PITX1, activates Pomc transcription (70). PAX7 controls melanotroph differentiation (71) and facilitates TBX19-controlled Pomc transcription via chromatin remodeling (72).

DKO-increased Pomc expression in the pituitary gland was associated with increased hormone secretion in vivo and in vitro. Serum beta-endorphin was significantly elevated in both DKO females and males ( Figure 2D ), as were serum concentrations of alpha-melanocyte stimulating hormone ( Figure 2E ) and adrenocorticotropic hormone ( Figure 2F ). In cultured pituitary cells, both hormone release and cellular content of these hormones were significantly elevated, further indicating that Ptprn and Ptprn2 regulate their synthesis ad release. DKO also increased serum corticosterone concentration, adrenal mass, and gene expression of the steroidogenic enzyme Star in adrenal tissue in both sexes (69).

Elevated expression of Pax7 in the DKO pituitary is consistent with the hypothesis that Pomc expression and hormone synthesis and release are elevated in melanotrophs. The hypothesis was additionally confirmed by the finding that the expression of melanotroph-specific genes Pcsk2, Esm1, Doc2g, and Oacyl was also increased in DKO pituitaries. In contrast, there was no increase in the expression of the corticotrophs-specific genes Chrna1, Clrn1, Trdn, and Hspb3 (69). Finally, immunohistochemical analysis using a POMC/adrenocorticotropic hormone-specific antibody to identify corticotrophs and melanotrophs and PAX7-specific antibody to label melanotrophs, showed that the intermediate lobe (home to melanotrophs) was enlarged, reflecting an increase in the population size of DKO melanotrophs. In contrast, we failed to detect an increase in the number of corticotrophs in the anterior lobe ( Figure 2G ). Therefore, both melanotrophs. hyperplasia and increased Pomc expression per cell in DKO mice are responsible for the significant increase in POMC-derived hormones. The scheme shown in Figure 2H illustrates the interpretation of these findings.

A review of the PTPRN literature suggests an important conclusion; all neuroendocrine cells express PTPRN genes, but their knockout disrupts the function of only some of these cell types, suggesting a cell type-specific role for these pseudophosphatases. In the diffuse neuroendocrine system, the cells themselves control their own secretion, gene expression, and proliferation in response to stimulation. Thus, the role of PTPRNs in their function is more readily elucidated. In pancreatic β-cells, PTPRNs have been suggested to be an integral part of a system for monitoring β-cell-stimulated secretory activity and for adjusting insulin expression and initiating cell proliferation. There has also been significant progress in characterizing the molecular mechanism of action of PTPRNs in the exocytotic pathway, insulin gene expression, and cell proliferation. A growing number of reports also indicate that PTPRNs are involved in the tumorigenesis, but their mechanism of action has not been proven. The role of PTPRNs in other diffuse neuroendocrine cells has not been studied.

In the centralized neuroendocrine system organized as the hypothalamic-pituitary-target organ axes, there is a complex relationship as the participating cells synchronize their activity through feedforward and feedback mechanisms. It is therefore more difficult to identify the cell type directly affected by DKO, as demonstrated in work with the hypothalamic-pituitary-gonadal axis in female and male mice. Although PTPRNs are expressed in kisspeptinergic and GnRH neurons, as well as in gonadotrophs, only kisspeptinergic neurons in both regions of the hypothalamus have been identified as PTPRNs-responsive cell types, suggesting that stimulation of Kiss1 expression increases hormone gene expression and secretion downstream of the axis ( Figure 1E ). In contrast, in the hypothalamic-pituitary-adrenal axis, melanotrophs have been identified as directly responding cells. Moreover, PTPRNs appear to inhibit both Pomc expression and melanotroph proliferation/differentiation, in parallel with the action of dopamine ( Figure 2G ). Further studies are needed to characterize the role of PTPRNs in the function of other hypothalamic and pituitary cells.

It is known that transcription factors can act as activators and repressors in different cells, but it is currently unknown whether PTPRNs act as transcription factors or upstream elements in the control of gene transcription. Further studies are needed to elucidate the molecular mechanism of this process. The cellular specificity of the PTPRNs actions is consistent with the specificity of promoter activation and repression for different genes, as suggested by the failure of DKO to increase Pomc expression in the hypothalamus but facilitate Pomc expression in the pituitary. The role of PTPRNs in normal and carcinoma cell proliferation also requires further studies.