A calmodulin-dependent translocation pathway for small secretory proteins.

Metazoans secrete an extensive array of small proteins essential for intercellular communication, defense, and physiologic regulation. Their synthesis takes mere seconds, leaving minimal time for recognition by the machinery for cotranslational protein translocation into the ER. The pathway taken by these substrates to enter the ER is not known. Here, we show that both in vivo and in vitro, small secretory proteins can enter the ER posttranslationally via a transient cytosolic intermediate. This intermediate contained calmodulin selectively bound to the signal peptides of small secretory proteins. Calmodulin maintained the translocation competence of small-protein precursors, precluded their aggregation and degradation, and minimized their inappropriate interactions with other cytosolic polypeptide-binding proteins. Acute inhibition of calmodulin specifically impaired small-protein translocation in vitro and in cells. These findings establish a mammalian posttranslational pathway for small-protein secretion and identify an unexpected role for calmodulin in chaperoning these precursors safely through the cytosol.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

Prolactin levels in functional hypothalamic amenorrhea: a retrospective case-control study.

PURPOSE: Functional hypothalamic amenorrhea (FHA) is due to hypothalamic dysregulation. Literature lacks data about prolactin in FHA women, although both prolactin levels and FHA are associated with stress. Moreover, polycystic ovarian morphology is common in FHA and there is an association between FHA and polycystic ovary syndrome. Thus, the aim of this study was to assess prolactin levels in FHA patients and controls with a special focus on factors influencing prolactin levels, that could be considered as "sensors" of the hypothalamic-pituitary dysregulation. METHODS: In a retrospective cohort study, 140 women with clearly defined FHA were compared to 70 healthy, normally ovulating women matched for age. The main outcome parameter was prolactin. Factors associated with prolactin levels > 12 µg/L were tested using a multivariable binary logistic regression model. RESULTS: The median prolactin level was 11.5 µg/L (interquartile range, IQR 7.5-14.4), which was similar to the control group (median 10.7, IQR 8.3-14.5; p = 0.065). Only two women had hyperprolactinemia (prolactin > 25 µg/L; 1.4%). In a multivariable binary logistic regression model eating disorder (odds ratio, OR 0.206; p = 0.040), excessive exercise (OR 0.280; p = 0.031) and TSH (OR 1.923; p = 0.020) were significantly associated with prolactin levels > 12 µg/L. CONCLUSION: Women with FHA have similar prolactin levels to healthy age-matched individuals. Eating disorders and excessive exercise where associated with prolactin levels < 12 µg/L, in contrast to TSH.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

A computational approach for explaining the effect of the prl gene polymorphism on prolactin structure and biological activity in Japanese quails.

Prolactin is a versatile hormone with multiple activities, including a negative control on egg production. This study was conducted to genotype all the coding portions of the prl gene using PCR-SSCP-sequencing, and to investigate the effects of amino acid substitutions of the prl gene on the structure and function of prolactin in quails using in silico approach. Though all genotyped exons exerted homogenous PCR-SSCP patterns, a total of 12 novel SNPs were detected in the investigated exons, including three SNPs in exon-1, 8 SNPs in exon-2, and one SNP in exon-4. Three adjacent missense SNPs were detected in exon-2, namely H69P, T70P, and S71F. Computational tools indicated obvious deleterious effects of T70P, with less extent to H69P and S71F on prolactin functions and activity, which may lead to limited participation of this hormone in the negative control of egg production. In conclusion, the introduction of in silico prediction has suggested an alternative solution for the breeders to evaluate the effect of each witnessed nsSNP in protein structure and function. The current study suggests three nsSNPs, T70P, T70P, and S71F as strong candidates for the negative effect on prolactin biological activity with a consequent reversal positive effect on egg productivity traits.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

In silico prediction of prolactin molecules as a tool for equine genomics reproduction.

The prolactin hormone is involved in several biological functions, although its main role resides on reproduction. As it interferes on fertility changes, studies focused on human health have established a linkage of this hormone to fertility losses. Regarding animal research, there is still a lack of information about the structure of prolactin. In case of horse breeding, prolactin has a particular influence; once there is an individualization of these animals and equines are known for presenting several reproductive disorders. As there is no molecular structure available for the prolactin hormone and receptor, we performed several bioinformatics analyses through prediction and refinement softwares, as well as manual modifications. Aiming to elucidate the first computational structure of both molecules and analyse structural and functional aspects related to these proteins, here we provide the first known equine model for prolactin and prolactin receptor, which obtained high global quality scores in diverse software's for quality assessment. QMEAN overall score obtained for ePrl was (- 4.09) and QMEANbrane for ePrlr was (- 8.45), which proves the structures' reliability. This study will implement another tool in equine genomics in order to give light to interactions of these molecules, structural and functional alterations and therefore help diagnosing fertility problems, contributing in the selection of a high genetic herd.

A novel type of prolactin expressed in the bullfrog pituitary specifically during the larval period.

Prolactin (PRL) is one of the major hormones that control amphibian metamorphosis. Recently, a PRL (PRL1B) gene that is different from the known PRL (PRL1A) gene has been found in the genomes of several amphibian species. In order to ascertain whether the PRL1B gene is expressed in the bullfrog (Rana catesbeiana) pituitary, cloning of cDNA encoding PRL1B in the pituitary of the premetamorphic bullfrog tadpole was attempted. The bullfrog PRL1B amino acid sequence predicted from the obtained cDNA showed 62% identity with those of Xenopus PRL1Bs that have been presumed from the genome sequences, whereas the sequence identity between bullfrog PRL1A and PRL1B was 48%. A molecular phylogenetic tree showed that bullfrog PRL1B is most appropriately grouped with amphibian PRL1Bs. Quantitative PCR analysis revealed that the mRNA expression levels of bullfrog PRL1B in the pituitary were high during pre- and prometamorphosis, sharply declined at metamorphic climax and became undetectable after metamorphosis. In contrast, PRL1A mRNA levels were relatively low during pre- and prometamorphosis, rose at climax and remained high after metamorphosis. Immunohistochemical study using antibodies against partial peptides of PRL1A and PRL1B revealed that most of the PRL1A- and PRL1B-immunoreactive cells in the larval pituitary were distributed separately, but that some of the cells immunoreactive with both antibodies were also present. Western blot analysis with the larval pituitary extract indicated that PRL1B-immunoreactive band appeared at the position of molecular weight ca. 22.1 kDa and PRL1A-immunoreactive band at the position of ca. 22.8 kDa. The results obtained in this experiment suggest the possibility that PRL1B plays as-yet-unknown role(s) during the pre-climactic period of metamorphosis. This is the first report on the existence of PRL1B as a protein in the amphibian larval pituitary.

Molecular evolution of prolactin in Chiroptera: Accelerated evolution and a large insertion in vespertilionid bats.

Pituitary prolactin (PRL) shows an episodic pattern of evolution in mammals, with a slow underlying rate (near stasis) and periods of rapid change in some groups. PRL evolution in bats, the second most speciose mammalian order, has not previously been studied, and is examined here. Slow basal evolution of PRL is seen in some bats, particularly megabats, but in most microbat groups evolution of PRL is more rapid. Accelerated evolution of PRL is particularly notable in the family Vespertilionidae, where analysis of nonsynonymous and synonymous substitutions indicates that it reflects adaptive evolution/positive selection. Remarkably, vespertilionid bats also show a large sequence insertion, of variable length, into exon 4 of PRL, giving a protein sequence 18-60 amino acids longer than normal, with the longest insertions in bats of the genus Myotis. An equivalent insertion has not been reported in PRL of any other vertebrate group. In the 3-dimensional structure of the complex between PRL and the extracellular domain (ecd) of its receptor (PRL:PRLR2) the inserted sequence is seen to be introduced in the short loop between helices 2 and 3 of PRL; it is far removed from the receptor-binding sites, and may not interfere with binding. The ecd of the receptor also shows variable rates of evolution, with a higher rate in the Vespertilionidae, but this is much less marked than for the hormone. The distribution of substitutions introduced into PRL during vespertilionid evolution appears to be non-random, and this and the evidence for positive selection suggests that the rapid evolution and insert sequence introduction were associated with a significant change in the biological properties of the hormone.

Protein targeting and degradation are coupled for elimination of mislocalized proteins.

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.

The effect of different protease inhibitors on stability of parathyroid hormone, insulin, and prolactin levels under different lag times and storage conditions until analysis.

INTRODUCTION: Proteolytic cleavage through proteases affects peptide hormone levels, which is of particular significance when the time interval between sampling and analysis is prolonged. We evaluated the stability of parathyroid hormone, insulin, and prolactin molecules (i) with different protease inhibitors such as K2 EDTA, aprotinin, and protease inhibitor cocktail (PIC), (ii) with different lag times (6-72 hours), and (iii) under different storage temperatures (4°C vs room temperature [RT]) until analysis. MATERIALS AND METHODS: Blood samples were collected into 2 sets of 5 Vacutainer® tubes (Becton Dickinson) from 10 healthy adults. Tubes 1 and 2 were plain gel separator tubes. Tubes 3, 4, and 5 contained PIC (1%), aprotinin (500 KIU/mL), and K2 EDTA, respectively. After centrifugation at 1300 g for 10 minutes, PIC added to tube 2 of each set. Samples were analyzed and then one set was stored at 4°C, whereas the other at RT until analysis at 6, 24, 48, and 72 hours. Hormone levels were determined with electrochemiluminescence immunoassay (ModularE170; Roche Diagnostics). The results were compared with desirable bias limits (DBL) from Westgard QC database. RESULTS: Insulin at RT decreases exceeding the DBL starting from 24 hours and K2 EDTA preserved insulin. PTH exceeded the DBL at RT for 48 hours or longer and PIC addition after centrifugation inhibited its degradation. Prolactin remained stable in all tested conditions. All parameters in the plain gel separator tubes remained within DBL when stored at 4°C until 72 hours. CONCLUSIONS: Different proteases may degrade peptide hormones and measures should be taken to counteract these effects especially if there is a delay before analysis.

Influence of sulpiride treatment on the level of prolactin and immunoglobulins in the peripheral blood of mares during the postpartum period.

The aim of this study was to determine the effect of increased levels of prolactin (PRL) on the concentration of immunoglobulins in the blood, colostrum and milk of mares. The study was conducted on 12 mares of the Polish Pony breed (6 in the control and 6 in the experimental group). To induce hyperprolactinaemia in mares of the experimental group, 750 mg sulpiride was administered orally once a day. The initial PRL concentration was 52.22 ± 11.21 ng/ml in the control group and 49.39 ± 10.12 ng/ml in the experimental group. In the subsequent days, the concentration of PRL dynamically changed. Statistical analysis showed highly significant differences (P < 0.01) between the groups. The concentration of immunoglobulins in the blood plasma was at the same level during the experimental period (32.97-29.08 mg/ml in the experimental group and 28.60-18.11 mg/ml in the control group). Statistical analysis showed highly significant differences between the groups in blood plasma immunoglobulin level (P < 0.01). The highest immunoglobulin concentration was obtained within 12 h after parturition in the control and the experimental group (23.49 ± 2.12 mg/ml and 26.94 ±1.72 mg/ml, respectively). The lowest values were obtained on day 12 after parturition in the experimental group (10.15 mg/ml ± 1.47 mg/ml) and on day 7 after parturition in the control group (14.30 mg/ml ± 2.48 mg/ml). In conclusion, this study did not provide evidence that the lactogenic hormone prolactin is involved in the transfer of immunoglobulins into the colostrum in horses.

A Complex Dance: The Importance of Glycosaminoglycans and Zinc in the Aggregation of Human Prolactin.

The zinc binding hormone pituitary human prolactin (hPRL) is stored in secretory granules of specialized cells in an aggregated form. Glycosaminoglycans (GAGs) are anionic polysaccharides commonly associated with secretory granules, indicating their involvement in granule formation. Here we, for the first time, study the impact of GAGs in combination with Zn(2+) on the reversible hPRL aggregation across the pH range of 7.4-5.5. Zn(2+) alone causes hPRL aggregation at pH 7.4, while aggregation between pH 7.4 and 5.5 requires both Zn(2+) and GAGs. GAGs alone cause hPRL aggregation below pH 5.5. Comprehensive thermal stability investigations show that hPRL is particularly destabilized toward thermal denaturation at pH 5.5 and that GAGs increasingly destabilize hPRL at decreasing pH values. We propose that Zn(2+) causes hPRL aggregation through low-affinity Zn(2+) binding sites on hPRL with GAGs facilitating Zn(2+) binding by neutralizing repulsive positive charges of hPRL in the acidic environments of the TGN and mature secretory granules. In a manner independent of the aggregation-causing agent(s), the different hPRL aggregates show very similar secondary structure and amorphous morphology. We speculate that this may be a recognizable sorting signal in the formation of hPRL granular vesicles.

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin.

BACKGROUND: Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function. METHODS: Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches. RESULTS: Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers. CONCLUSIONS: These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function. GENERAL SIGNIFICANCE: Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure-function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.

Urine oligosaccharide pattern in patients with hyperprolactinaemia.

Free milk-type oligosaccharides are produced during pregnancy and lactation and may have an impact on several cells in the immune system. Our aim was to investigate if patients with isolated hyperprolactinaemia, not related to pregnancy, also have increased synthesis and urinary excretion of milk-type oligosaccharides and to compare the excretion pattern with that found during pregnancy. Urine samples were collected as morning sample from 18 patients with hyperprolactinaemia, 13 healthy controls with normal prolactin levels and four pregnant women. After purification, lactose and free oligosaccharides were analysed and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. The identity of peaks was confirmed by exoglycosidase treatment and comparison with oligosaccharide standards. Prolactin was measured in serum collected between 09 and 11 a.m. by a standardized immunochemical method. Patients with hyperprolactinaemia had higher urinary excretion of lactose than normoprolactinemic controls and urinary lactose correlated positively to prolactin levels (r = 0.51, p < 0.05). Increased levels of the fucosylated oligosaccharides 2-fucosyl lactose and lacto-di-fucotetraose were found in urine from three and two patients, respectively. The acidic oligosaccharide 3-sialyl lactose was found in high amount in urine from two patients with prolactin of >10,000 mU/l. However, pregnant women in their third trimester had the highest concentration of all these oligosaccharides and excretion increased during pregnancy. This study is first to show that both lactose and certain fucosylated and sialylated milk-type oligosaccharides are increased in some patients with hyperprolactinaemia. It remains to elucidate the functional importance of these findings.

Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

BACKGROUND: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response. FINDINGS: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway. CONCLUSIONS: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

Effects of age and reproductive experience on the distribution of prolactin and growth hormone secreting cells in the anterior pituitary of a passerine.

Plasma prolactin (PRL) is released from lactotrophs in the anterior pituitary. As plasma PRL levels rise during incubation in domestic fowl, the number of lactotrophs (PRL-immunoreactive, PRL-IR cells) increases while the number of growth hormone secreting cells, somatotrophs (GH-IR cells), declines. We measured plasma PRL levels using radioimmunoassay (RIA) and examined the distribution of lactotrophs and somatotrophs in the anterior pituitary of breeding and nonbreeding zebra finches of known ages with and without prior breeding experience using fluorescent immunohistochemistry (IHC). Plasma PRL levels were higher in breeding than in nonbreeding birds, regardless of age, sex, or previous breeding history. PRL-IR cells were localized primarily, but not exclusively, to the cephalic aspect of the anterior pituitary (AP) and along the ventral margin. Birds with prior reproductive experience had more PRL-IR cells than birds with no prior reproductive experience and breeders had slightly higher PRL-IR cell counts than did nonbreeders, but there was no correlation between the number of PRL-IR cells and plasma PRL levels. GH-IR cells were concentrated in the caudal aspect of the AP with some cells in the cephalic lobe, but numbers did not differ between any of the groups studied. An increase in PRL-IR cells corresponded with an increase in GH-IR cells. An increase in lactotroph number with reproductive experience in zebra finches may facilitate future reproductive events by allowing for more robust PRL secretion and increased reproductive success.

The many faces of prolactin in breast cancer.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

Ligand-mimicking receptor variant discloses binding and activation mode of prolactin-releasing peptide.

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.