No abstract

Background: DNA-protein cross-links (DPCs) are formed by DNA-damaging agents. Results: DPCs on the translocating strand but not on the nontranslocating strand block hexameric replicative helicases in a size-dependent manner. Stalled helicases dissociate from DNA with a half-life of 15-36 min. Conclusion: DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. Significance: Reversible and irreversible protein roadblocks may have distinct effects on replisomes.

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.

Melanin-concentrating hormone receptor 1 (MCHR1) is a G protein-coupled receptor (GPCR) highly expressed in the central nervous system. MCHR1 mediates many physiological functions including energy homeostasis and emotional processing. By acting as GTPase-activating proteins, regulators of G protein-signaling (RGS) proteins are negative modulators of GPCRs. We previously elucidated that RGS8 of the B/R4 RGS subfamily potently inhibits the action of both Galphaq- and Galphai/o-dependent MCHR1 signaling. In the present study of living cells, we provide evidence that another B/R4 protein, RGS2, is an efficient regulator of MCHR1-mediated calcium signaling exclusively via the Galphaq-dependent pathway. This effect was not observed for RGS4 and RGS5 proteins. Cotransfection of RGS2 with RGS8 additively increased the potency for inhibition of MCHR1 signaling. Truncation experiments revealed that an internal sequence within the N-terminal region of RGS2 (amino acids 28-80) was involved in the RGS2 modulation of MCHR1 activity. Our data suggest that RGS2 and RGS8 differentially associate with MCHR1 and may represent two distinct modes of signaling mechanisms in vivo.