Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A2B receptors of native human and rodent cell lines were investigated using [3H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [3H]PSB-298 showed saturable and reversible binding. It exhibited a KD value of 60 ± 1 nM and limited capacity (Bmax = 3.511 fmol per milligram protein) at recombinant human adenosine A2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A2B receptors. Adenosine A2B receptors were shown to be the major adenosine A2 receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [3H]PSB-298 is a selective radioligand for adenosine A2B binding sites in this cell line

Mapping of calmodulin and G beta gamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A

Ca2+/calmodulin (Ca2+/CaM) and the subunits of heterotrimeric G-proteins (G) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and G binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca2+/CaM. Mutational analysis of the Ca2+/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic -helix. Substitutions adjacent to the core CaM target sequence selectively prevent G binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating G recruitment. In addition, we present evidence that G uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although G-mediated signaling is abolished in receptors lacking the core CaM binding sequence, subunit activation, as assayed by agonist-dependent GTPS binding, was not affected. This suggests that Ca2+/CaM may alter the mode of group III mGluR signaling from mono- () to bidirectional ( and ) activation of downstream effector cascades. <br/