William F. Simonds

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism–jaw tumor (HPT–JT) syndrome. A single locus associated with HPT–JT (HRPT2) was previously mapped to chromosomal region 1q25–q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT–JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT–JT and in development of some sporadic parathyroid tumors.

G protein regulation of adenylate cyclase

Adenylate cyclase integrates positive and negative signals that act through G protein-coupled cell-surface receptors with other extracellular stimuli to finely regulate levels of cAMP within the cell. Recently, the structures of the cyclase catalytic core complexed with the plant diterpene forskolin, and a cyclase-forskolin complex bound to an activated form of the stimulatory G protein subunit Gs alpha have been solved by X-ray crystallography. These structures provide a wealth of detail about how different signals could converge at the core cyclase domains to regulate catalysis. In this article, William Simonds reviews recent advances in the molecular and structural biology of this key regulatory enzyme, which provide new insight into its ability to integrate multiple signals in diverse cellular contexts.

Familial isolated hyperparathyroidism: clinical and genetic characteristics of 36 kindreds.

Familial hyperparathyroidism (HPT) encompasses a clinically and genetically heterogeneous group of disorders. Syndromes with familial HPT include multiple endocrine neoplasia type 1 (MEN1) (Mendelian Inheritance in Man [MIM] 1311001) (63, 87), multiple endocrine neoplasia type 2A (MEN2A) (MIM 171400)(42, 79, 86), familial hypocalciuric hypercalcemia (FHH) (MIM 145980, 145981, 600740) also known as familial benign hypercalcemia (38, 64), and the hyperparathyroidism-jaw tumor syndrome (HPT-JT; HRPT2) (MIM 145001) (45). Familial isolated hyperparathyroidism2 (FIH; HRPT1) (MIM 145000) is a subgroup of familial HPT that can result from the incomplete expression of a syndromic form of familial HPT or from full expression of other entities (Figure 1). It is unknown how many as yet unrecognized clinical entities, including mutant genotypes, can also present as FIH. MEN1 is an autosomal dominant disorder characterized by endocrine and nonendocrine tumors, most strikingly involving the parathyroids, enteropancreatic endocrine system, and pituitary. Because FIH is seen less frequently than full expressions of MEN1, because HPT is the earliest and most frequent endocrinopathy in MEN1, and because even some large families with an apparent phenotype of FIH ultimately express MEN1, we (59, 67) and others (2, 61) previously speculated that most kindreds with FIH were occult expressions of MEN1. The gene responsible for MEN1 has been cloned (15), leading to powerful gene sequencing methods applicable to MEN1, FIH, and other conditions (63). MEN2A, unlike MEN1, is not typically a consideration in the differential diagnosis of FIH, because the higher penetrance of medullary thyroid carcinoma and pheochromocytoma than of HPT in MEN2A dominates the clinical presentation in a family (42, 79, 86). FHH is an autosomal dominant trait usually causing mild HPT (62) with relative hypocalciuria; hypercalcemia in FHH is highly penetrant at all ages, even in the perinatal period (64). Mild hypermagnesemia is sometimes seen in FHH but is unusual in other forms of primary HPT (53, 64). FHH cases almost always remain hypercalcemic following standard subtotal parathyroidectomy (PTX) (64). FHH always presents 0025-7974/02/8101-0001/0 MEDICINE® 81: 1-26, 2002 Vol. 81, No. 1 Copyright

Multiple endocrine neoplasia type 1: new clinical and basic findings

Multiple endocrine neoplasia type 1 (MEN1) provides a prime example of how a rare disease can advance our understanding of basic cell biology, neoplasia and common endocrine tumors. MEN1 is expressed mainly as parathyroid, enteropancreatic neuroendocrine, anterior pituitary and foregut carcinoid tumors. It is an autosomal dominant disease caused by mutation of the MEN1 gene. Since its identification, the MEN1 gene has been implicated in many common endocrine and non-endocrine tumors. This is a brief overview of recent scientific advances relating to MEN1, including newly recognized clinical features that are now better characterized by genetic analysis, insights into the function of the MEN1-encoded protein menin, and refined recommendations for mutation testing and tumor screening, which highlight our increasing understanding of this complex syndrome.

Molecular Pathology of the MEN1 Gene

Abstract: Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1‐like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF‐kB, PEM, SMAD3, RPA2, FANCD2, NM23β, nonmuscle myosin heavy chain II‐A, GFAP, and/or vimentin. These partners have not clarified menins pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun‐kinase pathway. The Men1+/− mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.

Parafibromin, product of the hyperparathyroidism-jaw tumor syndrome gene HRPT2, regulates cyclin D1/PRAD1 expression

Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism–jaw tumor familial cancer syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism. Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex. This study addressed the expression and functional properties of human parafibromin. A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle. Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments. Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas. Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia. These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.

Receptor and effector interactions of Gs Functional studies with antibodies to the αs carboxyl‐terminal decapeptide

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl‐terminus of Gs‐α have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild‐type S49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc − S49 cells, which lack Gs. RM blocks receptor‐mediated activation of Gs and adenylyl cyclase in membranes from wild‐type S49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[γ]S‐ or fluoride‐preactivated bovine brain membranes; thus binding of αs to effector and carboxyl‐terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G‐proteins with receptors and electors in the membrane.

The G protein connection: molecular basis of membrane association

Two distinct types of lipid modification, myristoylation and isoprenylation, are critical for membrane association of heterotrimeric G proteins. Elucidation of the molecular basis for G protein membrane association has important implications for understanding G protein structure and function, and is relevant to potential therapeutic approaches to AIDS and cancer.

SELECTIVE ACTIVATION OF EFFECTOR PATHWAYS BY BRAIN-SPECIFIC G PROTEIN BETA5

While multiple G protein β and γ subunit isoforms have been identified, the implications of this potential diversity of βγ heterodimers for signaling through βγ-regulated effector pathways remains unclear. Furthermore the molecular mechanism(s) by which the βγ complex modulates diverse mammalian effector molecules is unknown. Effector signaling by the structurally distinct brain-specific β5 subunit was assessed by transient cotransfection with γ2 in COS cells and compared with β1. Transfection of either β1 or β5 with γ2 stimulated the activity of cotransfected phospholipase C-β2 (PLC-β2), as previously reported. In contrast, cotransfection of β1 but not β5 with γ2 stimulated the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways even though the expression of β5 in COS cells was evident by immunoblotting. The G protein β5 expressed in transfected COS cells was properly folded as its pattern of stable C-terminal proteolytic fragments was identical to that of native brain β5. The inability of β5 to activate the MAPK and JNK pathways was not overcome by cotransfection with three additional Gγ isoforms. These results suggest it is the Gβ subunit which determines the pattern of downstream signaling by the βγ complex and imply that the structural features of the βγ complex mediating effector regulation may differ among effectors.

Differential Modulation of Adenylyl Cyclases I and II by Various Gβ Subunits

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by Gβγ dimers states that the various isoforms of AC respond differently to the presence of free Gβγ. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by Gβγ subunits. This result does not address the possible differences in modulation that may exist among the different Gβγ heterodimers. Six isoforms of Gβ and 12 isoforms of Gγ have been cloned to date. We have established a cell transfection system in which Gβ and Gγ cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both Gβ1/γ2 and Gβ5/γ2 combinations, AC II responded differentially and was stimulated by Gβ1/γ2 and inhibited by Gβ5/γ2. This finding demonstrates differential modulatory activity by different combinations of Gβγ on the same AC isoform and demonstrates another level of complexity within the AC signaling system.