The cyclin-dependent kinase inhibitor p16 is a candidate tumour-suppressor protein that maps to a genomic locus strongly associated with familial melanoma and other tumour types. Screening of primary tumours and linkage analysis of familial melanoma pedigrees have identified many potential mutations in p16, but the functional significance of these sequence variants has remained unclear. We report here that p16 can act as a potent and specific inhibitor of progression through the G1 phase of the cell cycle, and we demonstrate that several tumour-derived alleles of p16 encode functionally compromised proteins. The ability of p16 to arrest cell-cycle progression generally correlates with inhibition of cyclin D1/Cdk4 kinase activity in vitro, with two exceptions among the alleles tested. In vivo, the presence of functional retinoblastoma protein appears to be necessary but may not be sufficient to confer full sensitivity to p16-mediated growth arrest. Our results provide support for the notion that p16 is an important cell-cycle regulator whose inactivation contributes to the outgrowth of human tumours.

The effects of transforming growth factor ␤ (TGF-␤) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1 S , and 184A1L5 R , which differ in their cell cycle responses to TGF-␤ but express type I and type II TGF-␤ receptors and retain TGF-␤ induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-␤ was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-␤-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p15 INK4B protein stability. As cdk4-and cdk6-associated p15 INK4B increased during TGF-␤ arrest of sensitive cells, there was a loss of cyclin D1, p21 Cip1 , and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15 INK4B complexes did not contain detectable cyclin. p15 INK4Bfrom both sensitive and resistant cells could displace in vitro cyclin D1, p21 Cip1, and p27 Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5 R cell lysates. Thus, in TGF-␤ arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-␤-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-␤ is underlined by the observation that in TGF-␤-resistant 184A1L5 R , although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.Cell cycle transitions are governed by a family of cyclindependent kinases (cdks), whose activity is regulated by association with positive effectors, the cyclins (41, 61), with negative regulators, the cdk inhibitors (62), and by phosphorylation (45,66). The cdks control a series of biochemical pathways, or checkpoints, which integrate extracellular mitogenic and growth-inhibitory signals, monitor chromosome integrity, and coordinate cell cycle transitions (23, 42). Passage through G 1 into S phase is regulated by the activities of cyclin D-, cyclin E-, and cyclin A-associated kinases. D-type cdks play a major role in phosphorylation of the retinoblastoma protein (pRb) (61), which is required for G 1 -to-S phase transition.Two different families of cdk-inhibitory proteins have been identified (for reviews see references 58 and 62). The kinase inhibitor protein (KIP) family is composed of three members, p21 (also identified as Cip1, Waf1, and Sdi1), p27 (Kip1), and p57 (Kip2). p21 (21, 73) is a transcriptional target of p53 (9) and plays a role in senescence (44) and differentiation (17,49,64) and in the coordination of DNA damage repair with cell cycle arrest (6,13,34,52,72). p27Kip1 was identified as an inhibitory activity in cells arrested by TGF-␤ (see below) (31,55,56,65) and was cloned independe...

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclindependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in pl9ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve Po0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at Po0.05 (>8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with !5 somatic mutations also likely indicate functionally critical residues, even though P>0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

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Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.

Significance Parathyroid adenomas, the main cause of primary hyperparathyroidism (PHPT), are thought to result from clonal expansion of tumor cells and to be insensitive to normal calcium feedback due to the loss of the calcium-sensing receptor (CASR). Utilizing flow cytometric analysis to isolate and individually study oxyphil cells, chief cells, and lymphocytes from resected parathyroid tumors and glands, we now report previously unrecognized heterogeneity in these tissues with respect to calcium responsiveness, CASR expression, and clonal origin of parathyroid tumors. Such heterogeneity of parathyroid adenomas likely reflects the complex etiopathogenesis and clinical heterogeneity of PHPT.