To purify the renal V2 receptor and identify domains involved in hormone binding, photoaffinity labeling of the membrane-bound bovine V2 receptor with a tritium-labeled photoreactive vasopressin agonist was performed. The labeled 30,000 M(r) protein was purified to homogeneity by anion-exchange chromatography, isoelectric focusing, gel filtration, gel electrophoresis, and reversed-phase HPLC. N-terminal sequencing showed that the isolated protein which contains the covalently bound hormonal ligand, represents an N-terminal truncated bovine V2 receptor. The purified labeled V2 vasopressin receptor protein was digested with V8 protease, and peptide fragments were isolated. Protein microsequencing and comparison with the cDNA sequence of a cloned PCR product identified two extra- and two intracellular peptides of the bovine V2 receptor. Radioactivity was incorporated into two amino acid residues localized in the second extracellular domain. Our results indicate that this extracellular domain is involved in peptide agonist binding of the V2 receptor.
At the locus for arylsulfatase A (ASA) at least four to five alleles exist: besides the normal ASA+ and at least two to three deficiency alleles (ASA-), a pseudodeficiency allele, ASAp, is known. On SDS-PAGE the ASAp enzyme migrates slightly faster than ASA+. Treatment of extracts from cells with ASA+/ASA+, ASAp/ASAp, or ASA+/ASAp genotypes with endoglycosidase F leads to the same deglycosylated subunit pattern. Presumably the degree of glycosylation is lower in ASAp than in ASA+. In a large-scale screening project we determined a gene frequency of 7.3% for ASAp. Thus, the ASA locus is polymorphic. In seven families, ASAp showed a codominant mode of inheritance with ASA+. Homozygosity for ASAp has no obvious clinical consequences. In subjects with the compound genotype ASA-/ASAp, the residual enzyme activity may fall below a critical threshold, so that the substrate can no longer be hydrolyzed sufficiently. Since these compounds are not so rare (estimated frequency 0.073%), this mechanism could be of importance in neuropsychiatric disorders with late onset.
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