Allen M. Spiegel

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators.

•u2002 Introduction •u2002 The CaR is a unique family 3 GPCR •u2002 Architecture of the CaR ‐u2002 Venus flytrap domain ‐u2002 Cysteine‐rich domain ‐u2002 Seven‐transmembrane domain ‐u2002 Intracellular carboxyl terminus •u2002 Naturally occurring inactivating mutations associated with FHH and NSHPT •u2002 Naturally occurring activating mutations associated with ADH and Bartters syndrome type V •u2002 Allosteric modulators of the CaR and their therapeutic potentials ‐u2002 Positive allosteric modulators ‐u2002 Negative allosteric modulators •u2002 Concluding remarks

Changes in the guanine nucleotide-binding proteins, Gi and go, during differentiation of 3T3-L1 cells

Differentiation of 3T3‐L1 cells from fibroblasts to adipocytes is accompanied by increased adenylate cyclase response to lipolytic agents. We used pertussis toxin and specific antibodies to measure the inhibitory guanine nucleotide‐binding protein, Gi, and the novel G‐protein, Go, in membranes from 3T3‐L1 cells. Pertussis toxin‐dependent labeling of a 39–40 kDa protein showed an initial 30% rise, followed by an 80% fall during differentiation. Immunoblots showed that 3T3‐L1 cells contain Go, as well as Gi, and that changes in the former parallel the changes in pertussis toxin labeling. Changes in Gi and Go may contribute to altered adenylate cyclase response during 3T3‐L1 cell differentiation.

The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

'Personalized Medicine' To Identify Genetic Risks For Type 2 Diabetes And Focus Prevention: Can It Fulfill Its Promise?

Public health measures are required to address the worldwide increase in type 2 diabetes. Proponents of personalized medicine predict a future in which disease treatment and, more important, prevention will be tailored to high-risk individuals rather than populations and will be based on genetic and other new biomarker tests. Accurate biomarker tests to identify people at risk for diabetes could allow more-targeted and perhaps individualized prevention efforts. DNA variants conferring higher risk for type 2 diabetes have been identified. However, these account for only a small fraction of genetic risk, which limits their practical predictive value. Nor has identification of these variants yet led to new, individualized prevention methods. Further research is needed to identify genomic and other types of biomarkers that could accurately predict risk and facilitate targeted prevention.

Inherited Endocrine Diseases Involving G Proteins and G Protein-Coupled Receptors

Naturally occurring mutations in the G protein Gs-alpha subunit and in a number of G protein-coupled receptors (GPCRs) have been identified in human diseases. Loss-of-function mutations in GPCRs for various hormones lead to hormone resistance manifest as hypofunction of the gland expressing the affected GPCR. Conversely, GPCR gain-of-function mutations lead to hormone-independent activation and hyperfunction of the involved gland. Our laboratory has focused on the extracellular calcium-sensing GPCR (CaR) expressed primarily, but not exclusively, in parathyroid glands and kidney. Loss-of-function CaR mutations lead to a form of hyperparathyroidism, an apparent exception to the general pattern described above, but in fact reflecting resistance to the normal inhibition of parathyroid hormone secretion by the hormone agonist, extracellular Ca2+. CaR gain-of function-mutations cause autosomal dominant hypocalcemia due to activation of the receptor at subphysiologic concentrations of serum Ca2+, leading to inappropriate inhibition of parathyroid hormone secretion. I will describe our recent work that helps inform design of novel therapeutics targeting this important GPCR.

Future Diagnostic and Therapeutic Trends in Endocrine Cancers

Current treatment of endocrine cancers relies primarily on surgical resection, which is generally effective only for localized disease. Radioactive iodine treatment is an important modality for those thyroid cancers that maintain the ability to take up iodine. For endocrine cancers that are no longer localized, current modes of therapy, including various combinations of chemotherapy and radiation, are inadequate, posing a major challenge to ongoing research to develop more effective methods for diagnosis and treatment. In this article, we offer some predictions of future trends in the diagnosis and treatment of endocrine cancers. Following a general introduction, we focus on thyroid cancer as a paradigm for what we may expect in future developments, and then add selected comments relevant to parathyroid, adrenocortical, and gastrointestinal and pancreatic neuroendocrine tumors. Rapid, inexpensive whole genome sequencing of both germline and tumor DNA, novel molecular and functional imaging, as well as new biomarkers are expected to enable more precise diagnosis, targeted therapy, and possibly prevention. Translating the coming wave of data on the molecular pathogenesis of endocrine cancers into practical diagnostic and treatment modalities will require new forms of collaboration between investigators, clinicians, and industry.

G s α, Pseudohypoparathyroidism, Fibrous Dysplasia, and McCune–Albright Syndrome

G s α is a ubiquitously expressed heterotrimeric G protein α-subunit that couples numerous heptahelical receptors to stimulation of cAMP formation. Heterozygous, germline inactivating G s α mutations cause the congenital syndrome Albright hereditary osteodystrophy (AHO). Mutations on the maternal allele result in AHO plus multihormone resistance (pseudohypoparathyroidism type 1A, PHP1A) while those on the paternal allele result in the AHO phenotype alone (pseudoPHP). Imprinting defects in the G s α gene GNAS lead to a more isolated form of renal parthyroid hormone (PTH) resistance (PHP1B). Somatic gain-of-function mutations in G s α lead to constitutive cAMP formation. When such mutations occur in a highly localized manner, they can cause monostotic fibrous dysplasia or isolated endocrine tumors. Mutations occurring much earlier in development lead to a mosaic distribution of mutation-bearing cells, and phenotypic manifestations including polyostotic fibrous dysplasia, hyperfunction of multiple endocrine organs, cafe-au-lait skin pigmentation (all three together in McCune–Albright syndrome), and dysfunction of other organs.

Targeted therapies: Good news for advanced-stage pancreatic neuroendocrine tumors

A phase III, randomized, placebo-controlled trial in 171 patients with advanced-stage pancreatic neuroendocrine tumors demonstrated a significant improvement in progression-free survival with sunitinib treatment. We discuss the results of this trial in the context of other studies that have assessed treatment of pancreatic neuroendocrine tumors.

Following in the Footsteps of a “Neuroscience” Giant

Isaac Newton famously said in his 1675 letter to Robert Hooke: “If I have seen further it is by standing on the shoulders of giants.” As I look forward to the prospect of becoming Dean of the Albert Einstein College of Medicine (AECOM) on June 1, 2006, it is not so much a question of standing on the shoulders, but rather attempting to fill the enormously large shoes, of the giant who for the past 22 years has served as Dean of AECOM. Beginning with his publication, while still a medical student, of a series of papers on the electrical activity of single neurons, and continuing throughout his distinguished career as an investigator authoring landmark papers on the anatomy, development and physiology of the nervous system, Dominick Purpura virtually defined and founded the field of neuroscience.