Identification and functional characterization of novel calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia

Familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcemia (ADH), in which calcium homeostasis is disordered, are associated with mutations in the calcium-sensing receptor (CASR). Six unrelated kindreds with FHH and/or NSHPT and two unrelated kindreds with ADH were studied. Direct sequence analysis of the exons of the CASR gene identified heterozygous mutations in six of the kindreds with FHH and in one of those with ADH. We performed functional analyses on the novel missense and insertion/frameshift mutants by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. All mutant receptors were expressed at a similar level to that of the wild type; however, whereas mutants R220W and A835T (the ADH mutant) were fully glycosylated and were visualized on the cell surface, glycosylation of mutants G549R and C850-851 ins/fs was impaired, resulting in reduced cell surface staining. In fura-2-loaded HEK293 cells expressing the wild-type or mutant receptors, the inactivating R220W mutant produced a significant shift to the right relative to the wild-type CASR in the cytosolic calcium response to increasing extracellular calcium concentrations and the G549R and C850-851 ins/fs mutants were without detectable activity. The activating A835T mutation resulted in a shift to the left in the cytosolic calcium response to extracellular calcium concentrations relative to the wild type. Our studies have identified novel CASR mutations that alter the function of the CASR in several different ways.     

Cyclic AMP phosphodiesterases in human lymphocytes

The function of lymphocytes, like platelets, has been shown to be inhibited by agents which increase intracellular cyclic AMP. Two high-affinity cAMP phosphodiesterases (PDEs), the cyclic GMP-inhibited cAMP phosphodiesterase, PDE3, and the cAMP-specific phosphodiesterase PDE4, are known to regulate cAMP concentration in haemopoietic cells by degrading cAMP to AMP. We characterized the relative contribution of the two PDEs to total lymphocyte PDE activity. We then determined which of the different gene products, PDE3A, typical of myocardium and platelets, or PDE3B, typical of adipocytes, were present in lymphocytes. The PDE3-specific inhibitor, milrinone, and the PDE4 inhibitor, rolipram, suppressed hydrolysis by 70% and 30% respectively, which indicated that both PDE4 and PDE3 were present, and that PDE3 was predominant. RT-PCR yields the expected size fragment for the primer pair PDE3B and not for PDE3A. The DNA sequence obtained had > 95% identity with PDE3B. PDE3B appears to be the major cAMP PDE in lymphocytes. In contrast to human platelets, human lymphocytes appear to contain the PDE3B subtype. Since PDE3B in adipocytes is subject to hormonal regulation, lymphocytes may be similarly modulated. Understanding the role of cAMP regulation and the involvement of cAMP in lymphocyte function may have important implications in drug development.     

D2 dopamine receptor-induced sensitization of adenylyl cyclase type 1 is G alpha(s) independent

Acute activation of D2 dopamine receptors inhibits adenylyl cyclase (EC 4.6.1.1), whereas persistent activation of these inhibitory receptors results in a compensatory increase in cyclic AMP accumulation. This sensitization of adenylyl cyclase is thought to involve enhanced Galpha(s)-adenylyl cyclase interactions; however, the absolute requirement of Galpha(s) has not been determined. The present study used a Galpha(s)-deficient cell line to examine directly the role of Galpha(s) in D2 dopamine receptor-induced sensitization of recombinant adenylyl cyclase type 1 (AC1) and 5 (AC5). In acute experiments, quinpirole activation of the D2 dopamine receptor inhibited AC1 and AC5 activity, indicating that the acute regulatory properties of AC1 and AC5 were retained in the absence of Galpha(s). Subsequent experiments revealed that short-term (2 h) activation of the D2 dopamine receptor resulted in significantly enhanced forskolin-stimulated AC1 activity in the absence of Galpha(s), whereas sensitization of forskolin-stimulated AC5 activity appeared to require Galpha(s). The Galpha(s)-independent sensitization of AC1 was explored further using AC1-selective activation protocols (A23187 and CCE) following short- and long-term agonist treatment. These studies revealed that persistent activation of D2 dopamine receptors sensitized AC1 activity to Ca2+ stimulation in cells devoid of endogenous Galpha(s) and demonstrate directly that sensitization of AC1 is Galpha(s)-independent.     

The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3

Hypocalcemia and hyperphosphatemia caused by parathyroid hormone (PTH)-resistance are the only discernible abnormalities in pseudohypoparathyroidism type Ib (PHP-Ib). Because mutations in the PTH/PTH-related peptide receptor, a plausible candidate gene, had been excluded previously, we conducted a genome-wide search with four PHP-Ib kindreds and established linkage to a small telomeric region on chromosome 20q, which contains the stimulatory G protein gene. We, furthermore, showed that the genetic defect is imprinted paternally and thus is inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or pseudo-pseudohypoparathyroidism, two related disorders caused by different stimulatory G protein mutations.     

Identification of a region of the C-terminal domain involved in short-term desensitization of the prostaglandin EP4 receptor

1. The prostaglandin EP4 receptor, which couples to stimulation of adenylyl cyclase, undergoes rapid agonist-induced desensitization when expressed in CHO-K1 cells. 2. Truncation of the 488-amino acid receptor at residue 350 removes the carboxy-terminal domain and abolishes desensitization. 3. To further delineate residues involved in desensitization, the receptor was truncated at position 408, 383 or 369. Receptors truncated at position 408 or 383 underwent PGE2-induced desensitization, whereas the receptor truncated at position 369 displayed sustained activity, indicating that the essential residues for desensitization lie between 370 and 383. 4. The six serines in the 14-amino acid segment between residues 370 and 383 were mutated to alanine, retaining the entire C-terminal domain. Desensitization was absent in cells expressing this mutant. 5. The results indicate involvement of serines located between 370 and 382 in rapid desensitization of the EP4 receptor.     

Molecular diagnosis of pseudohypoparathyroidism type Ib in a family with presumed paroxysmal dyskinesia

We describe 2 sisters diagnosed initially with paroxysmal kinesigenic choreoathetosis, a condition characterized by brief episodes of spasms precipitated by sudden movement. However, subsequent testing showed hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone levels consistent with pseudohypoparathyroidism type Ib. This diagnosis was confirmed by genetic testing, which identified a 3-kilobase deletion on chromosome 20q13.3. Our report describes the neurologic presentation, metabolic derangement, and underlying genetic mutation in a family. It also reinforces the importance of metabolic testing in the evaluation of pediatric patients with movement disorders.     

Inherited hypophosphatemic disorders in children and the evolving mechanisms of phosphate regulation

Phosphorous is essential for multiple cellular functions and constitutes an important mineral in bone. Hypophosphatemia in children leads to rickets resulting in abnormal growth and often skeletal deformities. Among various causes of low serum phosphorous are inherited disorders associated with increased urinary excretion of phosphate, including autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemia (XLH), autosomal recessive hypophosphatemia (ARHP), and hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Recent genetic analyses and subsequent biochemical and animal studies have revealed several novel molecules that appear to play key roles in the regulation of renal phosphate handling. These include a protein with abundant expression in bone, fibroblast growth factor 23 (FGF23), which has proven to be a circulating hormone that inhibits tubular reabsorption of phosphate in the kidney. Two other bone-specific proteins, PHEX and dentin matrix protein 1 (DMP1), appear to be necessary for limiting the expression of fibroblast growth factor 23, thereby allowing sufficient renal conservation of phosphate. This review focuses on the clinical, biochemical, and genetic features of inherited hypophosphatemic disorders, and presents the current understanding of hormonal and molecular mechanisms that govern phosphorous homeostasis.