Table 2:

Binding affinity of PTH(1–34) with divalent ion concentrations

Mg2+		Ca2+
Concentration (mM)	KD (nM)	Concentration (mM)	KD (nM)
0	1132 ± 59	0	1132 ± 59
3	862 ± 36	5	200 ± 18
10	600 ± 52	10	114 ±19
15	732 ± 23	15	146 ± 6
40	650 ± 65	40	61 ± 4
100	628 ± 24	100	105 ± 6

Cell based cAMP assays. Ligand binding to PTH1R activates G protein signaling cascades, leading to the production of cAMP. To determine if the calcium dependent binding of PTH(1–34) affects the activation of PTH1R, we performed cAMP assays in the presence and absence of 15 mM Ca²⁺ (Figure 5). The addition of 15 mM Ca²⁺ did not affect the potency of PTH(1–34) in activating PTH1R. However, 15 mM Ca²⁺ significantly decreased the amount of cAMP produced by PTH(1–34) activation of PTH1R. Control experiments to test the effect of 15 mM Ca²⁺ on cAMP production in the absence of PTH(1–34) show that the cAMP produced decreases from 6.8 ± 2.6 to 0.8 ± 0.2 pmol/mg total protein upon addition of 15 mM Ca²⁺. Upon PTH(1–34) stimulation of PTH1R, the maximum cAMP produced was 276 ± 67 and 154 ± 42 pmol/mg total protein in the absence and presence of 15 mM Ca²⁺, respectively. These results suggest 15 mM Ca²⁺ decreases PTH(1–34) activation of PTH1R; however, it is still important to consider a portion of this decrease could be due to the effect of 15 mM Ca²⁺ alone on cAMP production of the cells.