Parathyroid cells express a cell surface receptor, coupled to the mobilization of intracellular Ca2+, that is activated by increases in the concentration of extracellular Ca2+ and by a variety of other cations. This "Ca2+ receptor" (CaR) serves as the primary physiological regulator of parathyroid hormone secretion. Alterations in the CaR have been proposed to underlie the increases in Ca2+ set-point seen in primary hyperparathyroidism due to parathyroid adenoma. We have isolated human CaR cDNAs from an adenomatous parathyroid gland. The cloned receptor, expressed in Xenopus oocytes, responds to extracellular application of physiologically relevant concentrations of Ca2+ and other CaR agonists. The rank order of potency of CaR agonists displayed by the native receptor (Gd3+ > neomycin B > Ca2+ > Mg2+) is maintained by the expressed receptor. The nucleotide sequence of the human CaR cDNA predicts a protein of 1078 amino acids with high sequence similarity to a bovine CaR, and displays seven putative membrane-spanning regions common to G protein-coupled receptors. The deduced protein sequence shows potential sites for N-linked glycosylation and phosphorylation by protein kinase C and has a low level of sequence similarity to the metabotropic glutamate receptors. Comparison of the cDNA sequence to that of the normal human CaR gene showed no alteration in the coding region sequence of the CaR in this particular instance of parathyroid adenoma. Human cDNA clones with differing 5'-untranslated regions were isolated, suggesting alternative splicing of the parathyroid CaR mRNA. A rare variant cDNA clone representing a 10 amino acid insertion into the extracellular domain was also isolated. Northern blot analysis of normal and adenomatous parathyroid gland mRNA identified a predominant transcript of approximately 5.4 kilobases, and less abundant transcripts of approximately 10, 4.8 and 4.2 kilobases in RNA from the adenoma. While there is no evidence for alteration of the primary amino acid sequence of the CaR in this adenoma, modulation of CaR biosynthesis through alternative RNA processing may play a role in set-point alterations.

The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+-induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH-terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2-terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH.

Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands. Fifteen of these scFv-phage, identified using IL-1R(I)-binding assays and dissociation rate ranking, were reformatted as scFv-Fc and IgG(4) molecules. The ease of producing antibodies in the scFv-Fc format permitted rapid identification of four lead clones (C10, C13, C14, C15) that inhibit NF-kappaB nuclear translocation induced by IL-1. Reformatting these clones as IgG(4) molecules increased their inhibition potency by

Reportedly, most acromegalics are refractory to the growth hormone (GH)-releasing effect of central nervous system-acting stimuli. For instance, the synthetic analogue of met-enkephalin (Enk) viz. FK 33-824 fails to alter the high circulating GH levels of acromegalics. The most likely interpretation of such finding is that circulating GH disrupts, for a negative feedback effect, hypothalamic opioid function and/or GH-releasing hormone (GHRH) producing neurons, through which opioids exert their action. To address this issue, we have evaluated in intact and hypophysectomized male rats the effect of a high-dose GH regimen on the hypothalamic stores of endogenous opioid peptides, beta-endorphin (beta-EP) and met-enkephalin (met-enk). Moreover we have evaluated in intact male rats the effect of exogenous GH on median eminence (ME) GHRH stores and the ability of FK 33-824 to stimulate GH and prolactin (PRL) secretion and of exogenous GHRH to induce GH secretion. Human GH (25 and 250 micrograms bid for 4 days) administered to hypophysectomized rats strikingly reduced beta-EP and met-enk-like immunoreactivity (LI) in the medial basal hypothalamus, the effect being already maximal with the lower hGH dose. The higher dose of hGH diminished, though to a lower extent, hypothalamic beta EP-LI content also in intact rats, and reduced GHRH-LI content in the ME. Despite these profound biochemical alterations, the GH responsiveness to GHRH and FK 33-824 administration was preserved, while the latter drug induced a lower PRL rise in GH-treated than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)