The intrinsic property of cell-penetrating peptides (CPPs) to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be potential components of future drugs and disease diagnostic agents. These versatile peptides are simple to synthesize, functionalize, and characterize yet are able to deliver covalently or noncovalently conjugated bioactive cargos (from small chemical drugs to large plasmid DNA) inside cells, primarily via endocytosis, in order to obtain high levels of gene expression, gene silencing, or tumor targeting. Typically, CPPs are often passive and nonselective yet must be functionalized or chemically modified to create effective delivery vectors that succeed in targeting specific cells or tissues. Furthermore, the design of clinically effective systemic delivery systems requires the same amount of attention to detail in both design of the delivered cargo and the cell-penetrating peptide used to deliver it.

GPCR135, publicly known as somatostatin-and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35 S-labeled guanosine 5-O-(3-thiotriphosphate) (GTP␥S) incorporation in cells overexpressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTP␥S binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC 50 values of 0.25 and 0.35 nM, respectively.125 I-Relaxin-3 binds GPCR135 at high affinity with a K d value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.The recent completion of the sequencing of the human genome revealed thousands of new genes. Among them are many orphan G-protein-coupled receptors (GPCRs), 1 which are identified from genomic DNA or mRNA sequences based on their predicted seven-transmembrane structures. Searching for ligands of the orphan GPCRs has because been an intense research area and has yielded numerous significant discoveries in the past decade (1-15). Identification of ligand/receptor pairs provides a basis for the understanding of the physiological roles of those GPCRs and their ligands, which can involve the central nervous, endocrine, reproductive, cardiovascular, immune, inflammatory, digestive, and metabolic systems (1-15). The identification of ligands for their receptors also provides additional opportunities to discover agonists and antagonists as innovative drugs to exert pharmacological effects by interacting with these newly identified receptors.Relaxin is a member of the insulin superfamily. The hallmark of this protein family is the presence of two peptide subunits that are arranged by three disulfide bonds (16 -19). Whereas insulin is known to play a major role in glucose metabolism and signals through the insulin receptor, a single transmembrane growth factor/tyr...

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of 125 I-relaxin-3 to GPCR142 with a high affinity (K i ‫؍‬ 1.5 nM). In a saturation binding assay, 125 The relaxin/insulin family peptides include insulin (1), IGF1 1 (2), IGF2 (3), relaxin (4, 5), INSL3 (6), INSL4 (7), INSL5 (8), INSL6 (9), and relaxin-3/INSL7 (10). Except for IGF1 and IGF2, which are single chain peptides, each member of the family consists of two peptide subunits (an A-chain and a B-chain) that are linked by three disulfide bonds (4 -14). Insulin, IGF1, and IGF2 are known to be involved in the regulation of glucose metabolism (15) and signal through tyrosine kinase/ growth factor receptors, which are single transmembrane receptors (16, 17). Relaxin plays multifunctional roles including uterus relaxation, reproductive tissue growth, and collagen remodeling in females (18). In addition, relaxin has been reported to play important roles in nonreproductive functions including wound healing, cardiac protection, and allergic responses (19). The receptor for relaxin has been identified recently as a leucine-rich repeat containing the G-protein-coupled receptor (LGR) LGR7 (20). Although relaxin also activates LGR8 in vitro (20), recent studies show that LGR8 is likely the endogenous receptor for INSL3 and is involved in testis descent (21,22). To date, the receptors for INSL4, INSL5, and INSL6 have not been identified. Relaxin-3 (also known as INSL7), the most recently identified member of the family, was reported to be an additional ligand for LGR7 (23). We recently identified relaxin-3 as a ligand for two orphan G-protein-coupled receptors GPCR135 (14) and GPCR142 (24). The predominant brain expression for both 14,25, 26) and GPCR135 (14,26), coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135. The tissue expression pattern of GPCR142 (also known as GPR100), which is primarily in peripheral tissues (24), is drastically different from that of relaxin-3, suggesting that GPCR142 may have an endogenous ligand other than relaxin-3. Furthermore, despite the high conservation of relaxin-3 in different species, GPCR142 is less conserved in the mouse and is a pseudogene in the rat (26), suggesting that GPCR142 may have a diminished role in rodents and may function as a receptor for a different ligand (other than relaxin-3) in other mammals. Sequence analysis among insulin/relaxin family members indicates that INSL5 shares high homology to relaxin-3 (Fig. 1A), suggesting that it may be an additional ligand for GPCR135, GPCR142, LGR7, or LGR8. In this report, we demonstrate that INSL5 is an agonist for GPCR142 but not for GPCR135, LGR7, or LGR8.

Relaxin-3 has recently been identified as a ligand for two structurally related G-protein-coupled receptors, human GPCR135 and GPCR142. This current study reports the characterization of mouse and rat GPCR135 as well as GPCR142 from mouse, monkey, cow, and pig at the molecular and pharmacological levels. Mouse and rat GPCR135 exhibit high homology (Ͼ85%) to the human GPCR135 and have very similar pharmacological properties to that of the human GPCR135. Human and mouse/ rat relaxin-3 both bind to and activate mouse, rat, and human GPCR135 at high affinity with IC 50 or EC 50 values close to 0.5 nM. In contrast, the mouse GPCR142 is less well conserved (74% homology) with human GPCR142. The rat GPCR142 gene was found to be a pseudogene. We further cloned GPCR142 genes from monkey, cow, and pig and found that they are highly homologous (Ͼ84%) to human GPCR142. Pharmacological characterization of GPCR142 from different species demonstrated that relaxin-3 binds to GPCR142 from different species at high affinity (IC 50 Ͻ 5 nM). However, relaxin-3 does not stimulate a Ca 2ϩ response in cells coexpressing G␣ 16 and mouse GPCR142, whereas it does for cells expressing GPCR142 from other species tested. Our results suggest that GPCR142 may have a diminished role as a receptor for relaxin-3 in rodents, or perhaps GPCR142 functions as a receptor for another ligand in nonrodents. Boels and Schaller recently reported bradykinin as a ligand for GPCR142 (also known as GPR100). In this report, we demonstrate that bradykinin activates neither GPCR135 nor GPCR142, whereas relaxin-3 does.

Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1β, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 μM and 5 μM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.