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.
Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. ␣-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related ␣3 and ␣6 subunits. Structure-function analysis of ␣-conotoxin MII was performed in an attempt to generate analogs with preference for ␣6-containing [␣6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at ␣3* versus ␣6* nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. 125 I]␣-conotoxin MII binding to putative ␣62* nAChRs in mouse brain homogenates (K i ϭ 3.3 nM). Thus, structure-function analysis of ␣-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing ␣3 and ␣6 subunits.nAChRs activated by the endogenous neurotransmitter acetylcholine belong to the superfamily of ligand-gated ion channels that also includes GABA A , 5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). These different ligand-gated ion channels show considerable sequence and structural homology. Each of the subunits has a relatively hydrophilic amino terminal half (ϳ200 amino acids) that constitutes an extracellular domain. This is followed by three hydrophobic transmembrane domains, a large intracellular loop, and then a fourth hydrophobic transmembrane span.A large number of genes have been cloned that encode subunits of nAChRs. It has been proposed that these subunits may be divided into subfamilies on the basis of both gene structure and mature protein sequence. The subunits ␣2, ␣3, ␣4, and ␣6 belong to subfamily III, tribe 1; 2 and 4 belong to tribe III-2; and the putative structural subunits ␣5 and 3 belong to tribe III-3 (Corringer et al., 2000). Within tribe III-1, subunits ␣3 and ␣6 show considerable sequence identity (ϳ80% in the ligand-binding extracellular domain). Thus, designing ligands to distinguish between ␣3* 1 and ␣6* is particularly challenging.␣-Conotoxin MII is a 16 amino acid peptide originally isolated from the venom of the marine snail Conus magus. This peptide potently targets neuronal in preference to the muscle subtype of nicotinic receptor with high affinity for both ␣32 and ␣6* nAChRs. Unfortunately, ␣-conotoxin MII may not distinguish well between ␣3* and ␣6* nAChRs (Kuryatov et al., 2000). In an effort to remedy this situation and produce a selective ligand for ␣6* nAChRs, we have generated a series of ␣-conotoxin MII analogs.The ␣6 subunit is expressed in catecholaminergic neurons and in retina (Le Novère et al., 1996Vailati et al., 1999). In striatum, ␣6* nAChRs seem to play a central role in the modulation ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.