The expanding repertoire of receptor activity modifying protein (RAMP) function

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B, and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention

Adrenomedullin and endocrine control of immune cells during pregnancy

The immunology of pregnancy is complex and incompletely understood. Aberrant immune activity in the decidua and in the placenta is believed to play a role in diseases of pregnancy, such as infertility, miscarriage, fetal growth restriction and preeclampsia. Here, we briefly review the endocrine control of uterine natural killer cell populations and their functions by the peptide hormone adrenomedullin. Studies in genetic animal models have revealed the critical importance of adrenomedullin dosage at the maternal–fetal interface, with cells from both the maternal and fetal compartments contributing to essential aspects underlying appropriate uterine receptivity, implantation and vascular remodeling of spiral arteries. These basic insights into the crosstalk between the endocrine and immune systems within the maternal–fetal interface may ultimately translate to a better understanding of the functions and consequences of dysregulated adrenomedullin levels in clinically complicated pregnancies

Uterine natural killer cells as modulators of the maternal-fetal vasculature

Precise and local control of the innate immune system within the placenta is an essential component for achieving a normal and healthy pregnancy. One of the most abundant immune cells of the placenta is a subpopulation of natural killer (NK) cells that profusely populates the uterine decidua during early pregnancy. Uterine NK (uNK) cells and trophoblast cells of the placenta communicate both directly and indirectly to contribute to the critical process of spiral artery remodeling. Here, we discuss recent findings that expand our knowledge of uNK cell-trophoblast cell crosstalk and the important role it plays in the maternal vascular adaptation to pregnancy

Sumo-dependent substrate targeting of the SUMO protease Ulp1

<p>Abstract</p> <p>Background</p> <p>In the yeast <it>Saccharomyces cerevisiae</it>, the essential small ubiquitin-like modifier (SUMO) protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood.</p> <p>Results</p> <p>Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3)^(C580S), that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3)^(C580S)to purify Smt3-modified proteins from cell extracts.</p> <p>Conclusions</p> <p>Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates <it>in vivo </it>and <it>in vitro</it>. Furthermore, we found that a substrate-trapping Ulp1(3)^(C580S)interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.</p

Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females

A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase

Factors Influencing Graduate Program Choice Among Undergraduate Women

Context: Despite equal enrollment proportions in MD and PhD programs, there are fewer women than men in MD-PhD programs and academic medicine. Factors important in degree program selection, including the perception of gender disparities, among undergraduate students were characterized. Methods: In 2017, pre-health students at four public North Carolina universities were invited to participate in an online survey regarding career plans, decision factors, and perceptions of gender disparities in MD, PhD and MD-PhD pathways. The authors characterized factors important to program selection, and evaluated the association of intended graduate program with perceived gender disparities using Fisher’s exact tests. Results: Among the n=186 female survey participants, most were white (54%) and intended MD, PhD, and/or MD-PhD programs (52%). Sixty percent had heard of MD-PhD programs, over half had no research experience, and half were considering but uncertain about pursuing a research career. The most common factors influencing degree program choice were perceived competitiveness as an applicant, desired future work environment, and desire for patient interaction. Twenty-five percent of students considering MD, PhD, and MD-PhD programs stated that perceived gender disparities during training for those degrees will influence their choice of program, however intended degree was not statistically associated with perceived gender disparities. Discussion: Perceived gender disparities may influence choice of graduate training program but are not among the top factors. Perceived competitiveness as an applicant is an important career consideration among undergraduate women. Strategies to increase awareness of MD-PhD programs, to encourage women to consider all training paths for which they are qualified are needed. Keywords: Education, Graduate; Sexism; Career Choice; Biomedical Research/education; Female What is known: Though men and women are nearly equally represented in MD-only and PhD-only programs, women are underrepresented in MD-PhD programs, which train physician-scientists. Prior studies have shown gender is not associated with rates of attrition from MD-PhD programs or differences in academic preparation, research interest, or research experience, suggesting enrollment differences by gender may be due to fewer women applying to MD-PhD programs. Gender parity in the physician-scientist workforce is critical to equitably serving a diverse patient population. What this study adds: This study is the first to examine the role of gender disparities in the career choices of undergraduate women. Given the moderate familiarity with MD-PhD training and lack of research experience among respondents, increased awareness of MD-PhD programs and expanded research opportunities may help undergraduates make informed career choices. This may increase women MD-PhD applicants, creating a more balanced physician-scientist workforce to address the needs of patients from all backgrounds

Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation.

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females

Loss of <i>Ramp2</i> leads to a proliferation defect in the labyrinth layer.

<p>(A and B) Immunofluorescence for BrdU in e13.5 placentas. Scale bars, 500<b>μ</b>m. (C and D) Immunofluorescence for Ki67 in the labyrinth of e13.5 placentas. Scale bars, 500 μm. (E) Ratio of <i>Bax</i> to <i>Bcl2</i> mRNA expression normalized to <i>Gapdh</i> as measured by qRT-PCR (n≥5 placentas per genotype). lb, labyrinth; jz, junctional zone; dec, decidua.</p

<i>In situ</i> hybridization of <i>Ramp2</i> in e13.5 placentas reveals robust expression in the labyrinth layer.

<p>(A) <i>In situ</i> hybridization in both the fetal and maternal compartments of the wild type placenta (n = 4). Scale bar, 100 μm. (B) <i>In situ</i> hybridization in the labyrinth layer. Scale bar, 50 μm. Inset images in both panels show sense control probes. CP, chorionic plate; LB, labyrinth; SP, spongiotrophoblast cells; DEC, decidua. Arrows point to giant trophoblast cells.</p