Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Federally regulated institutional review boards: The aspects of a "model" IRB---Is the current system in need of a tune up or complete overhaul?

Institutional Review Boards (IRBs) are the primary gatekeepers for the protection of ethical standards of federally regulated research on human subjects in this country. This paper focuses on what general, broad measures that may be instituted or enhanced to exemplify a "model IRB". This is done by examining the current regulatory standards of federally regulated IRBs, not private or commercial boards, and how many of those standards have been found either inadequate or not generally understood or followed. The analysis includes suggestions on how to bring about changes in order to make the IRB process more efficient, less subject to litigation, and create standardized educational protocols for members. The paper also considers how to include better oversight for multi-center research, increased centralization of IRBs, utilization of Data Safety Monitoring Boards when necessary, payment for research protocol review, voluntary accreditation, and the institution of evaluation/quality assurance programs. ^ This is a policy study utilizing secondary analysis of publicly available data. Therefore, the research for this paper focuses on scholarly medical/legal journals, web information from the Department of Health and Human Services, Federal Drug Administration, and the Office of the Inspector General, Accreditation Programs, law review articles, and current regulations applicable to the relevant portions of the paper. ^ Two issues are found to be consistently cited by the literature as major concerns. One is a need for basic, standardized educational requirements across all IRBs and its members, and secondly, much stricter and more informed management of continuing research. There is no federally regulated formal education system currently in place for IRB members, except for certain NIH-based trials. Also, IRBs are not keeping up with research once a study has begun, and although regulated to do so, it does not appear to be a great priority. This is the area most in danger of increased litigation. Other issues such as voluntary accreditation and outcomes evaluation are slowing gaining steam as the processes are becoming more available and more sought after, such as JCAHO accrediting of hospitals. ^ Adopting the principles discussed in this paper should promote better use of a local IRBs time, money, and expertise for protecting the vulnerable population in their care. Without further improvements to the system, there is concern that private and commercial IRBs will attempt to create a monopoly on much of the clinical research in the future as they are not as heavily regulated and can therefore offer companies quicker and more convenient reviews. IRBs need to consider the advantages of charging for their unique and important services as a cost of doing business. More importantly, there must be a minimum standard of education for all IRB members in the area of the ethical standards of human research and a greater emphasis placed on the follow-up of ongoing research as this is the most critical time for study participants and may soon lead to the largest area for litigation. Additionally, there should be a centralized IRB for multi-site trials or a study website with important information affecting the trial in real time. There needs to be development of standards and metrics to assess the performance of the IRBs for quality assurance and outcome evaluations. The boards should not be content to run the business of human subjects' research without determining how well that function is actually being carried out. It is important that federally regulated IRBs provide excellence in human research and promote those values most important to the public at large.^