Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1.

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia.

We describe a patient with Blomstrand chondrodysplasia, a lethal genetic disorder characterized by extremely advanced endochondral bone maturation, in whom a homozygous missense mutation is present in the gene coding for the PTH/PTHrP receptor that leads to the substitution of a proline for a leucine in the N-terminal portion of the receptor (P132L). PTH-induced cAMP accumulation was severely reduced in COS-7 cells expressing P132L receptors compared to that of cells expressing wild-type receptors, and PTH-induced inositol phosphate accumulation was not detectable in cells expressing the mutant receptor. Similar results were obtained using PTHrP as an agonist. Maximal specific binding of radioiodinated [Tyr36]PTHrp(1-36) by cells transfected with the P132L receptor was < 10% of that observed for cells transfected with the wild-type receptor. Despite the reduction in radioligand binding to P132L receptors, the intensity and distribution of the fluorescent signal resulting from the expression of receptors fused to GFP were similar for cells transfected with the wild-type and mutant P132L receptors, suggesting a similar degree of cell surface expression. These results firmly establish the role of abnormalities in the PTH/PTHrP receptor in the pathogenesis of Blomstrand chondrodysplasia, and thereby confirm the importance of signaling through the PTH/PTHrP receptor in human fetal skeletal development. Because the amino-acid mutated in the patient described here is otherwise conserved in all mammalian class II G protein-coupled receptors, this abnormality may provide insights into structural features needed for the normal function of this family of receptors.

Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia.

We report the absence of functional parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors (PTH/PTHrP receptor) in Blomstrand chondrodysplasia, a genetic disorder characterized by advanced endochondral bone maturation. Analysis of PTH/PTHrP receptor genomic DNA from a patient with Blomstrand chondrodysplasia demonstrated that the patient was heterozygous for a point mutation (G--> A substitution at nucleotide 1176) inherited from the mother. Analysis of PTH/PTHrP receptor cDNA demonstrated that: (a) this point mutation caused the deletion of the first 11 amino acids of exon M5 (encoding the fifth transmembrane domain of the receptor), resulting from the use of a novel splice site created by the base substitution; (b) the mutant receptor was well expressed in COS-7 cells, but did not bind PTH or PTHrP, and failed to induce detectable stimulation of either cAMP or inositol phosphate production in response to these ligands; and (c) the paternal allele was not expressed. Thus, only the abnormal and nonfunctional PTH/PTHrP receptors encoded by the maternal allele were expressed by chondrocytes from this patient. In view of the known role played by the PTH/PTHrP receptor in bone and cartilage development, these results strongly support the conclusion that the absence of functional PTH/ PTHrP receptors is responsible for the skeletal abnormalities seen in Blomstrand chondrodysplasia, abnormalities that are the mirror image of those observed in Jansen's chondrodysplasia. These findings emphasize the importance of signaling through this receptor in human fetal skeletal development.

Parathyroid hormone-induced calcium release from intracellular stores in a human kidney cell line in the absence of stimulation of cyclic adenosine 3',5'-monophosphate production.

PTH-induced mobilization of cytosolic Ca2+ in a human kidney cell line (HEK/W) occurring in the absence of cAMP stimulation was characterized and compared with that obtained in the same cells stably transfected by the PTH/PTH-related peptide (PTHrp) receptor (HEK/T). In both cell lines, N-terminal fragments of PTH and PTHrp induced a concentration-dependent biphasic stimulation in [Ca2+]i: a transient peak followed by a slow linear increase. These increases in [Ca2+]i were inhibited by the PTH antagonist [Nle(8,18),Tyr(34)]bPTH(3-34). The transient peaks were due to calcium release from intracellular stores, as they resisted quenching of calcium in the extracellular buffer and were abolished by prior emptying of intracellular stores. These peaks differed, however, both in latency period and in magnitude, in the two cell lines. The phospholipase C inhibitor U73122 inhibited the PTH-induced increase in [Ca2+]i in HEK/T cells, but not in HEK/W. Similarly, PTH-induced inositol phosphate (InsPs) production was detected in HEK/T but not in HEK/W cells. PTH-induced calcium release in HEK/W cells was inhibited by the simultaneous presence of ryanodine and U73122. Low level PTH/PTHrp receptor messenger RNA expression was demonstrated by ribonuclease protection in HEK/W cells, although no specific binding of [125I]PTHrP(1-34) could be detected. Amplification products for the PTH/PTHrp receptor 1, but no other isoforms, were detected by RT-PCR in HEK/W cells. As expected, HEK/T cells responded to PTH by a 500-fold stimulation in cAMP production and expressed large numbers of PTH/PTHrp receptors, as shown by [125I]PTHrp binding. These results demonstrate that the signal transduction pathways activated by PTH in HEK/W and HEK/T cells are different. Because the major difference in these cell lines is the number of PTH/PTHrp receptors expressed, these results suggest that the transduction of signals by the PTH/PTHrp receptor is controlled by receptor number in such a way that PTH stimulates an increase in intracellular calcium in the absence of stimulation of InsPs and cAMP production in cells expressing low levels of PTH/PTHrp receptor, but stimulates calcium release through an InsPs pathway and induces cAMP production in cells expressing large numbers of PTH/PTHrp receptors. The control of receptor number may be one of the mechanisms through which PTH effects are regulated.

Expression of alternatively spliced isoforms of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger RNA in human kidney and bone cells.

Using a PCR-based strategy, two variants of the PTH/PTH-related peptide (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splicing of exons coding for the N-terminal portion of the receptor. In the S-N3-E2 isoform, the exon coding the signal peptide (S) is spliced to an alternative 3'-acceptor site, producing a product respecting the reading frame, but in which the E1 exon is replaced by 12 amino acids derived from the N3 intron. In the S-E2 isoform, in which the E1 exon is deleted by cassette exclusion, the reading frame is changed, but a truncated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese hamster ovary cells with the originally described S-E1-E2 isoform and the two splice variants, active transcription of PTH/PTH-rp receptor mRNA was detected by RT-PCR in all cases. Cell lines transfected with the S-E1-E2 and S-N3-E2 isoforms displayed a 15- to 25-fold and 2- to 3-fold increase, respectively, in cAMP content after stimulation with 2.4 x 10(-7) M human PTH(1-34), whereas cells transfected with the S-E2 isoform did not respond. PTH elicited an increase in intracellular calcium only in cells transfected with the S-E1-E2 isoform. Studies evaluating the surface expression of receptors using anti-human PTH/PTH-rp receptor antibodies and the ability of transfected cells to bind [125I]PTH-rp indicated that the low or absent responses to PTH stimulation resulted, at least in part, from low surface expression of the S-N3-E2 and S-E2 isoforms. These studies support the conclusion that exon E1 is extremely important in promoting surface expression of the PTH/PTH-rp receptor but indicate that isoforms lacking this exon can retain the ability to recognize PTH. The possible intracellular expression of these splice variants, which account for 15-20% of total PTH/PTH-rp receptor mRNA, needs to be evaluated.