Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

Evidence for an endogenous parathyroid hormone-sensitive adenylate cyclase activator

The middle medullary membrane of canine renal tissue was completely insensitive to parathyroid hormone (PTH) stimulation in the absence of GTP or tissue supernatant. In contrast, removal of endogenous GTP did not eliminate the PTH stimulation of cAMP formation in the renal cortical membrane preparation (P₃). Addition of boiled cortical P₃ to native cortical P₃ enhanced PTH stimulation in a dose-dependent manner. The boiled cortical P₃ was not active in middle medullary tissue. The minimum concentration of GTP which was required to cause stimulation in both cortical and middle medullary preparations was similar. The results suggest that (1) there are two classes of PTH-sensitive adenylate cyclase; one class is GTP dependent, while the other is GTP independent. (2) A new factor, as yet unidentified, in addition to GTP, is important in the regulation of adenylate cyclase by PTH in the renal cortex.     

Methionine acts as a "magnet" in photoaffinity crosslinking experiments

Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.