A role for JAK2 in mediating cell surface GHR-PRLR interaction

10 Growth hormone (GH) receptor (GHR) and prolactin (PRL) receptor (PRLR) are transmembrane class I 11 cytokine receptors that co-exist in various normal and cancerous cells. Both receptors respond to their 12 associated ligands predominantly by activating the Janus Kinase 2 (JAK2)-signal transducer and activator 13 of transcription (STAT) signaling pathways, and both are also known to initiate receptor-specific JAK2-14 independent signaling. Together with their cognate ligands, these receptors have been associated with 15 pro-tumorigenic effects in various cancers, including breast cancer (BC). Human GH is known to bind GHR 16 and PRLR, while PRL can only bind PRLR. A growing body of work suggests that GHR and PRLR can form 17 heteromers in BC cells, modulating GH signal transduction. However, the dynamics of PRLR and GHR on 18 the plasma membrane and how these could affect their respective signaling still need to be understood. 19 To this end, we set out to unravel the spatiotemporal dynamics of GHR and PRLR on the surface of human 20 T47D breast cancer cells and γ2A-JAK2 cells. We applied direct stochastic optical reconstruction 21 microscopy (dSTORM) and quantified the colocalization and availability of both receptors on the plasma 22 membrane at the nanometer scale at different time points following treatment with GH and PRL. In cells 23 co-expressing GHR and PRLR, we surprisingly observed that not only GH but also PRL treatment induces a 24 significant loss of surface GHR. In cells lacking PRLR or expressing a mutant PRLR deficient in JAK2 binding, 25 we observed that GH induces downregulation of membrane-bound GHR, but PRL no longer induces loss 26 of surface GHR. Colocalizations of GHR and PRLR were confirmed by proximity ligation (PL) assay. 27 Our results suggest that PRLR-GHR interaction, direct or indirect, is indispensable for PRL-but not GH-28 induced loss of surface GHR and for both GH-induced and PRL-induced increase of surface PRLR, with 29 potential consequences for downstream signaling. Furthermore, our results suggest that JAK2 binding via 30 the receptor intracellular domain’s Box1 element is crucial for the observed regulation of one class I 31 cytokine receptor’s cell surface availability via ligand-induced activation of another class I cytokine 32 receptor. Our findings shed new light on the reciprocal and collective role that PRLR and GHR play in 33 regulating cell signaling.

To this end, we set out to unravel the spatiotemporal dynamics of GHR and PRLR on the surface of human 20 T47D breast cancer cells and γ2A-JAK2 cells. We applied direct stochastic optical reconstruction 21 microscopy (dSTORM) and quantified the colocalization and availability of both receptors on the plasma 22 membrane at the nanometer scale at different time points following treatment with GH and PRL. In cells 23 co-expressing GHR and PRLR, we surprisingly observed that not only GH but also PRL treatment induces a 24 significant loss of surface GHR. In cells lacking PRLR or expressing a mutant PRLR deficient in JAK2 binding, 25 we observed that GH induces downregulation of membrane-bound GHR, but PRL no longer induces loss 26 of surface GHR. Colocalizations of GHR and PRLR were confirmed by proximity ligation (PL) assay. 27 Our results suggest that PRLR-GHR interaction, direct or indirect, is indispensable for PRL-but not  induced loss of surface GHR and for both GH-induced and PRL-induced increase of surface PRLR, with 29 potential consequences for downstream signaling. Furthermore, our results suggest that JAK2 binding via 30 the receptor intracellular domain's Box1 element is crucial for the observed regulation of one class I 31 cytokine receptor's cell surface availability via ligand-induced activation of another class I cytokine 32 receptor. Our findings shed new light on the reciprocal and collective role that PRLR and GHR play in 33 regulating cell signaling. 34 Introduction 38 Growth hormone (GH) and prolactin (PRL) are hormones emanating mainly from the anterior pituitary. 39 The primary function of GH is regulating anabolism and metabolism [1,2], while PRL has important roles 40 in breast development and lactation [3]. There is mounting evidence pointing at both hormones and their 41 receptors playing roles in various types of cancer [4][5][6][7][8][9][10][11], including breast cancer (BC) [12][13][14][15][16][17], where GHR 42 is frequently present and PRLR is often found overexpressed [18][19][20][21][22][23][24][25]. While they have been mostly 43 associated with pro-tumorigenic effects, PRL has also been reported to show anti-tumor effects and, like 44 PRLR, has been associated with good prognosis in certain BC subtypes [26][27][28][29]. However, a humanized 45 neutralizing monoclonal antibody directed against the extracellular domain of PRLR showed no anti-tumor 46 effect when administered in patients with PRLR-positive metastatic BC [30]. This suggests that PRLRs' pro-47 tumorigenic function may not be as relevant as previously thought or depends on other circumstances 48 such as the presence or absence of other hormone receptors, with which they may interact. 49 Both GH receptor (GHR) and PRL receptor (PRLR) are structurally similar transmembrane glycoproteins 50 and belong to the class I cytokine receptor superfamily [31,32]. GH can bind and introduce a 51 conformational change to both GHR and PRLR, allowing receptor activation and downstream signaling 52 [33][34][35][36][37], but, unlike GH, PRL can only bind to PRLR [36,[38][39][40]. Both GHR and PRLR lack intrinsic kinase 53 activity. However, as is characteristic of their superfamily members, both receptors contain a proline-rich 54 Box1 motif in the membrane-proximal region of their intracellular domains (ICDs). Following ligand 55 binding, downstream signal transduction involves predominantly activating the associated cytoplasmic 56 tyrosine kinase, Janus kinase 2 (JAK2), bound to the receptors' Box1 elements. This is followed by 57 phosphorylation of the signal transducer and activator of transcription 5 (STAT5) [41,42], although other 58 receptor-specific JAK2-independent signal transduction pathways may also be activated. 59 Increasing evidence indicates that GHR and PRLR interact. Two decades ago, it was shown that ovine GHR 60 (oGHR) and PRLR (oPRLR) can tightly associate with each other following stimulation with placental 61 lactogen [43]. These studies utilized chimeric receptors consisting of the extracellular domain (ECD) of 62 human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor (hGM-CSFR) along 63 with a part of either oGHR ICD or oPRLR ICD. After hGM-CSF treatment of cells co-expressing oGHR 64 chimera and oPRLR chimera, JAK2 was effectively activated, and protein-protein interaction of both 65 chimeric receptors was detected via co-immunoprecipitation [43,44]. Additionally, our previous work 66 revealed a specific ligand-independent human GHR (hGHR) -human PRLR ( distributed on the surface of T47D cells (Fig. 1). To assess the ligands' effects on the spatial distribution of 105 hGHR and hPRLR on the cell surface, we conducted time-course experiments using T47D cells with human 106 GH or human PRL (500 ng/ml each). Such stimulation is known to induce rapid and substantial STAT5 107 phosphorylation [45]. We first analyzed the cell surface localization density (number of localizations per 108 μm 2 ) of hGHR or hPRLR in both resting and ligand-stimulated conditions. The abundance of surface-hPRLR 109 was rapidly increased, reaching its maximum after 3 min of GH or PRL treatment with a ~5.6-fold and ~4.5-110 fold increase compared to the basal value, respectively ( Fig. 2A and 2B). This increase was followed by a 111 rapid decline: after GH treatment for 5 min, the localization density dropped from 200.6 ± 14.7 per μm 2 112 to 57.2 ± 3.6 per μm 2 ; after PRL treatment for 5 min, the density fell from 159.4 ± 16.2 per μm 2 to 94.8 ± 113 16.0 per μm 2 . In contrast to hPRLR, the density of hGHR significantly decreased from 43.3 ± 5.5 per μm 2 114 basally to 25.8 ± 2.7 per μm 2 after 3min of GH stimulation. After 5 min and 10 min of GH stimulation, the 115 density of hGHR remained relatively low at 26.6 ± 2.3 per μm 2 and 19.3 ± 1.5 per μm 2 , respectively ( Fig.  116  2C). Surprisingly, PRL, which does not bind to hGHR, also induced a loss of surface hGHR on T47D cells. 117 After 1 min of PRL treatment, only 36% of hGHR remained on the cell surface compared to the basal state, 118 and hGHR density remained low for at least 10 min (Fig. 2D). A schematic illustration of GH-and PRL-119 induced hGHR and hPRLR density changes is shown in Fig. 2E. We previously observed that hGHR and 120 hPRLR specifically co-immunoprecipitate in the absence of added ligands in T47D cells [45]; thus, we 121 postulate that the propensity of hGHR and hPRLR to physically interact underlies our observed loss of 122 hGHR in response to PRL stimulation. conjugated antibody (green, channel 1), and PRLR is labeled with Alexa 647-conjugated antibody (red, 128 channel 2). The last column of images shows the merging of these two channels. T47D cells were either 129 left untreated (upper row), exposed to 500ng/mL GH for 3 min (middle row), or exposed to 500ng/mL PRL 130 for 3 min (lower row). Brightness was increased by 40% and Contrast was reduced by 40% to increase 131 visibility. Scale bars 5μm. 132 3min.

GH or PRL stimulation induces a redistribution of hGHR and hPRLR clusters 142
We analyzed the dSTORM images using the DBSCAN algorithm to identify different clusters and determine 143 the number of receptor localizations within a cluster (termed 'cluster size') ( Fig. 3A). We then performed 144 a localization distribution analysis and plotted a histogram of the relative frequency of localizations 145 (termed 'distribution plot' in this study). Representative dSTORM images of hGHR as well as associated 146 distribution plots are shown in Fig. 3B. 147 To gain a better understanding of the changes in GHR-PRLR colocalizations, we obtained and analyzed the 148 corresponding bivariate cluster size distributions. Their median values are summarized in Fig. 3C and 3D. 149 As can be seen, hGHR responds quickly and transiently to GH stimulation by forming larger clusters. After 150 GH treatment for 1 min, the median number of hGHR blinking events in a cluster reaches its peak and 151 amounts to 85.6 ± 20.9 localizations in comparison with 33.3 ± 1.7 localizations per cluster in the basal 152 state (P value = 0.0035). After 5 min, this number is reduced approximately to its pre-stimulation value 153 (29.1 ± 2.2) but appears to continue to fluctuate for at least another 25 minutes (Fig. 3C). In turn, hPRLR 154 responds to GH in a less prominent manner: The median number of hPRLR blinking events in a cluster in 155 the basal state is 16.5 ± 0.7. After 3 min of GH stimulation, the median of PRLR cluster size reaches its 156 maximum, which is 22.5 ± 3.9 (P value = 0.1496), and afterward declines to its basal level (Fig. 3C). 157 Like the response to GH, hGHR reacts also quickly to PRL treatment. The median number of hGHR blinking 158 events in a cluster culminates at 1 min of PRL treatment with 45.8 ± 5.0 (compared with basal level, P 159 value = 0.0098). In distinction, hPRLR response to PRL is slower. The median number of hPRLR blinking 160 events in a cluster reaches its peak at 5 min of PRL treatment with a median number of 23.3 ± 2.8 161 (compared with basal level, P value = 0.0233), and after 10 min the median is only slightly less than that 162 maximum (Fig. 3D). Together, these results indicate that upon ligand stimulation hGHR cluster sizes 163 increase transiently and significantly, while changes of hPRLR cluster sizes occur slowly and more subtly. 164 Spatial proximity of hGHR and hPRLR upon ligand stimulation 176 Nanoscale interactions of hGHR and hPRLR on the cell surface have yet to be well established. To evaluate 177 the extent of hGHR and hPRLR surface colocalization on T47D cells, we utilized proximity ligation assays 178 (PLAs). In PLAs, a positive signal appears only when two target proteins are in proximity (<40 nm). Notably, 179 individual treatment with GH or PRL for 5 min decreased the PLA signal observed in untreated cells by 180 34.4% and 28.1%, respectively (Fig. 4A), suggesting either ligand caused a reduction in the total number 181 of colocalized hGHR and hPRLR clusters. To further validate these observations, we calculated the ratio of 182 colocalized clusters in dSTORM images. Treatment with GH or PRL for 1 min reduced the proportion of 183 colocalized clusters by nearly 50% (Fig. 4B and 4C). To analyze their compositions, we plotted 3D 184 distributions for the colocalized clusters after ligand treatments ( Fig. S1A and S1B). The probability of 185 observing co-localized clusters with numbers of hGHR and hPRLR that fall into certain bins is identified by 186 color and by the height of the bar on the z-axis. Following GH treatment for 1 min and 3 min, the number 187 of smallest clusters decreased, and the number of medium-sized clusters increased, suggesting a shift of 188 the bivariate distribution of co-localized cluster sizes toward medium-sized clusters (Fig. S1A). The 189 distribution of colocalized clusters after 5 min of GH treatment is similar to that at the basal state. In 190 contrast, after treatment with PRL for 3 min or more, the number of medium to large-sized clusters, 191 majoritarily containing either GHR or PRLR, decreased, while the number of smallest clusters increased 192 (Fig. S1B). Together, these results demonstrate that hGHR and hPRLR are spatially accessible to each other 193 and form receptor complexes on the T47D cell surface and that the nature of these complexes changes 194 differentially depending on the stimulating ligand. 195 expressing both hGHR and hPRLR. Thus, we sought to investigate the effect of PRL on hGHR in the absence 210 of hPRLR. We utilized CRISPR/Cas9 technology to generate hPRLR knockout T47D cells (termed T47D ΔPRLR ). 211

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In addition, to evaluate isolated hPRLR responses to ligands, we also generated hGHR knockout T47D cells 212 (termed T47D ΔGHR ). Immunoblot analysis with specific GHR and PRLR antibodies confirmed the absence of 213 hPRLR in T47D ΔPRLR cells and hGHR in T47D ΔGHR cells (Fig 5A). Similar to our results with parental T47D cells, 214 GH treatment of T47D ΔPRLR cells rapidly reduces the density of surface hGHR, suggesting that hPRLR need 215 not be present to allow this GH-induced effect (Fig 5B). However, contrary to findings in parental T47D 216 cells, PRL treatment of T47D ΔPRLR cells fails to modulate hGHR surface density (Fig 5C). Like parental T47D 217 cells, treatment of T47D ΔGHR cells with GH ( Fig 5D) or PRL (Fig 5E) yielded increased hPRLR surface 218 localizations. Thus, we conclude that the PRL-induced decrease of hGHR in T47D cells is dependent on the 219 presence of hPRLR, but the ability of both GH and PRL to increase surface hPRLR in T47D cells is 220 independent of hGHR's presence. 221 To extend our observations, we next examined the GH and PRL responses in a cellular reconstitution 222 system: γ2A-JAK2 [55-57] is a human JAK2-deficient fibrosarcoma cell line reconstituted with JAK2 that 223 stably expresses JAK2 but lacks hGHR and hPRLR. To independently study the role of each receptor in this 224 setting, we used our previously generated stable transfectants of γ2A-JAK2 cells that harbor either hGHR 225 or hPRLR [58] and verified the presence of the indicated receptor by immunoblotting (Fig. 5F). Consistent 226 with the observation in T47D ΔPRLR cells, γ2A-JAK2-hGHR cells responded with a loss of surface hGHR density 227 to GH stimulation but not to PRL stimulation (Fig. 5G). Interestingly, as in T47D ΔGHR cells, treatment of γ2A-228 JAK2-hPRLR cells with either GH or PRL (Fig. 5H) promotes increased surface hPRLR. Thus, our findings in 229 both cell systems suggest that hPRLR-hGHR interaction, direct or indirect, is indispensable for PRL-induced 230 but not for GH-induced loss of surface hGHR. The hPRLR-dependent PRL-induced hGHR downregulation indicates that hPRLR can modulate the density 250 of hGHR on the cell surface in response to PRL. To investigate this interaction, we generated a set of 251 truncation or deletion mutants of hPRLR: (1) hPRLR-tr292, which truncates the intracellular domain of 252 hPRLR distal to the membrane-proximal intracellular domain box 1 element; (2) hPRLR-tr238, which 253 contains only 4 amino acids of the proximal intracellular domain and does not include box 1; and (3)  254 hPRLR-Δbox1, in which the box 1 region (243aa-251aa) is internally deleted (shown in Fig. 6A). Expression 255 of hPRLR-tr292, hPRLR-tr238, and hPRLR-Δbox1, as well as wild-type hPRLR, was detected by 256 immunoblotting using monoclonal antibodies targeting the ECD of PRLR (mAb ext-1.48 ). Since hPRLR-tr238 257 contains only 4aa in its ICD, no immunoblot signal was detected using a polyclonal antibody (Ab AL-84 ) 258 targeting the hPRLR ICD. In contrast, the expression of hPRLR-tr292 was easily detected by Ab AL-84 (Fig. 6B). 259 We then transiently transfected each hPRLR construct into the γ2A-JAK2-hGHR cells, which stably express 260 hGHR, and analyzed the changes of cell surface hGHR localizations. In cells expressing wild-type hPRLR 261 (hPRLR-WT), the localizations of hGHR significantly decreased 3 min post-exposure to GH and PRL (Fig.  262 6C). Similarly, under the same treatment, in cells expressing hPRLR-tr292, the localization of hGHR on the 263 cell membrane was reduced in response to each ligand (Fig. 6D). However, in hPRLR-tr238-expressing cells, 264 the localization of hGHR was diminished by GH stimulation but slightly increased by PRL stimulation (Fig.  265 6E). To further investigate the role of the box1 region for hGHR and hPRLR functional interaction, we 266 studied hPRLR-Δbox1 expressing cells and found that upon PRL treatment, hGHR localizations did not 267 decrease on the cell surface (Fig. 6F). As a negative control, we transfected the cells with vector 268 (pcDNA3.1). hGHR localization decreased upon GH stimulation but remained at basal levels upon PRL 269 stimulation (Fig. 6G). In addition, in hPRLR-ΔBox1 and hGHR expressing cells, the JAK2 and STAT5 tyrosine 270 phosphorylation levels were assessed in response to GH and PRL stimulations. Treatment of 500ng/ml GH 271 induces a dramatic increase in both JAK2 and STAT5 phosphorylation. In contrast, 500ng/ml PRL treatment 272 does not cause JAK2 or STAT5 phosphorylation (Fig. 6H), consistent with PRLR Box1 being required for 273 effective coupling of PRL occupancy of PRLR to activation of JAK2 and phosphorylation of STAT5 and the 274 inability of PRL to signal via GHR. 275 Next, we analyzed colocalization in each group. In the resting state, the ratios of GHR-PRLR-colocalization 276 clusters are relatively higher in hPRLR-WT-and hPRLR-tr292-expressing cells in comparison with hPRLR-277 tr238-and hPRLR-ΔBox1-expressing cells (Fig. 6I). This suggests that the box 1 region in hPRLR plays a 278 critical role in stabilizing the hGHR-hPRLR complexes in the basal state. Detergent cell extracts of hPRLR-ΔBox1 and hGHR-expressing cells were analyzed by immunoblotting. 299 After 5 hrs. starvation, cells were treated with GH (500 ng/ml) or PRL (500 ng/ml) for 10 min. In each 300 experiment, the average Basal value is considered 100%. (I) Fraction of GHR/PRLR colocalized clusters. In 301 the resting state, the colocalization ratio is significantly lower for cells expressing hPRLR-tr238 than for 302 those expressing hPRLR-WT. Each data point represents the ratio of the number of clusters, which contain 303 both hGHR and hPRLR, to the total number of clusters on the cell surface. Data are displayed as mean ± 304 SE. * (P<0.05) indicates statistical significance in comparison with WT and is calculated by a two-tailed t-305 test assuming unequal variance (all bars without an asterisk are not significant). 306 307 Box 1 region in hGHR plays an essential role in regulating PRLR and GHR interaction 308 From our observations (Fig. 6), we concluded that the JAK2 binding site, i.e., the box 1 region, in hPRLR is 309 required for PRL-induced hGHR down-regulation from the cell surface. To further assess whether the JAK2 310 binding site on hGHR is also essential, we generated hGHR-ΔBox1, in which the box 1 region (297aa -311 305aa) was deleted (Fig. 7A). In cells expressing both hGHR-ΔBox1 and hPRLR we observed that GH does 312 not alter the hGHR-ΔBox1 localizations on the cell surface, while PRL slightly increases hGHR-ΔBox1 313 localizations (Fig. 7B). Together, these results re-affirm that binding of JAK2 to hGHR is also required for 314 hPRLR-mediated regulation of hGHR availability on the cell surface. 315 Cloning and constructs:

342
The human GHR cDNA in pcDNA1 was a generous gift from R. Ross (University of Sheffield, Sheffield, UK). 343 The human PRLR cDNA in pEF/V5/HIS was generously provided by C. Clevenger (Virginia Commonwealth 344

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University, Richmond, VA). hPRLR-tr238 and hPRLR-tr292 were generated by amplifying with external 345 primers containing EcorI site and a stop codon with XhoI site after the sequence of 238 or 292 amino acid, 346 respectively. hPRLR-ΔBox1, and hGHR-Δbox1 were generated by overlap extension polymerase chain 347 reaction (PCR) with associated primers and cloned into pcDNA3. images reveal that in human T47D breast cancer cells and the γ2A-JAK2 cell exogenous expression system, 399 the cluster size of hGHR and hPRLR in the basal state range from a few localizations per cluster up to a 400 thousand localizations per cluster. 401 With GH or PRL treatment, the number of hGHR on both T47D and γ2A-JAK2 cell surfaces is decreased, 402 indicating the removal of surface hGHR. Moreover, the distribution curve of hGHR shifts toward larger 403 cluster sizes (Fig. 3), suggesting a ligand-induced aggregation of receptors. In turn, hPRLR numbers 404 dramatically increase on the cell surface in response to ligand stimulation. The newly presented hPRLR 405 clusters may impact the distribution of cluster sizes, which may explain why the median of hPRLR cluster 406 sizes does not change much with ligand treatment. 407 Our PL assay data indicate that distances between hGHR and hPRLR are small (less than 40nm). This 408 implies that hGHR and hPRLR form co-localized clusters in unstimulated states, suggesting hGHR and 409 hPRLR are physically accessible to one other. With GH stimulation, the fraction of co-localized receptors 410 is decreased. Given that GH treatment enhances the coimmunoprecipitation of total cellular hGHR and 411 hPRLR [45], the decrease in co-localized surface receptors can likely be attributed to removing co-localized 412 receptors from the cell surface. 413 It is well documented that hPRLR is engaged by both PRL and GH, while hGHR only responds to GH [36,414 38,39]. Unexpectedly, we found that PRL induces a down-regulation of cell surface hGHR in cells that co-415 express hGHR and hPRLR (Fig. 2), indicating that hPRLR directly or indirectly interacts with hGHR. 416 Interestingly, PRL was unable to induce hGHR down-regulation in cells either lacking hPRLR or expressing 417 hPRLR without its Box1 region (hPRLR-tr238 or hPRLR-ΔBox1), suggesting that JAK2 and PRLR association 418 is required for hGHR-hPRLR interaction that in turn allows PRL-induced hGHR downregulation. Notably, 419 previous work showed that GHR and JAK2 association is necessary for JAK2 to stabilize cell surface GHR 420 and inhibit constitutive GHR down-regulation [57]. Furthermore, single-particle tracking studies showed 421 that, in the presence of JAK2, a higher level of ligand-induced dimerization of GHR was observed [68]. 422 Moreover, JAK2 with intact kinase activity is required for GH-induced GHR down-regulation [57]. Similarly, 423 the box1 region of PRLR also associates with JAK2, and deletion or modulation of the last proline residue 424 of box1 abrogates PRLR function [69]. Collectively, these findings suggest that the box1 regions in hGHR 425 and hPRLR play a crucial role in the transduction of the individual signaling cascades and hGHR-hPRLR 426 association. 427 Here we found that, in the resting state, the degree of hGHR-hPRLR colocalization is higher in cells 428 expressing hPRLR with the box1 motif. This suggests JAK2 may not only stabilize GHR but also support the 429 formation of hGHR-hPRLR-containing clusters. Previously, it has been suggested that such clusters are 430 comprised of hGHR homodimers and hPRLR homodimers that together form (hGHR-hGHR) -(hPRLR-431 hPRLR) hetero-multimers or higher order oligomers [46]. Indeed, because the intracellular domains (ICD) 432 of both hGHR and hPRLR are highly disordered, the flexibility of their ICDs may provide room for recruiting 433 JAK2 and stabilizing the hGHR-hPRLR association [70,71]. Moreover, a study of the crystal structure of 434 erythropoietin receptor (EPOR) and leptin receptor (LEPR), which also belong to the class I cytokine 435 receptor family, revealed recently that JAK2/EPOR and JAK2/LEPR complexes contained four JAK2 and 436 four EPOR or LEPR molecules, respectively [72]. Hence, it is possible that hGHR-hGHR homodimers and 437 hPRLR-hPRLR homodimers form complexes in a similar fashion (Fig. 8). We propose that this JAK2/Box1-438 mediated interaction of receptors is not limited to hGHR and hPRLR but may generalize to other receptors 439 of the class I cytokine receptor family. We note, however, that determinants within particular receptors 440 (perhaps residing in their extracellular and/or transmembrane domains) and their intracellular JAK2 441 association domains may facilitate, to varying degrees, their propensity to form multimeric aggregates. 442 GHR and PRLR, for example, may tend to do so more avidly with each other than either one does with 443 other cytokine receptors. Additional studies are required to determine and understand the modes and 444 functions of the hGHR-hPRLR association.